Methods for monitoring PCR amplification with fluorescently labeled probes or primers for which fluorescence changes upon hybridization to a target

ABSTRACT

A method is provided for determining a concentration of a target nucleic acid by using a nucleic acid probe labeled with a fluorescent dye. The method comprises:
         providing, as the probe, a nucleic acid probe capable of reducing fluorescence emission from the fluorescent dye when hybridized with the target nucleic acid;   hybridizing the probe to the target nucleic acid; and measuring a decrease in fluorescence emission from the fluorescent dye after the hybridization relative to fluorescence emission from the fluorescent dye before the hybridization.

The present application is a divisional application of U.S. applicationSer. No. 10/209,608 filed Aug. 1, 2002, now U.S. Pat. No. 7,094,540,which is a divisional application of U.S. Ser. No. 09/725,265, which wasfiled on Nov. 29, 2000, now U.S. Pat. No. 6,492,121; which is aDivisional Application of U.S. Ser. No. 09/556,127 filed Apr. 20, 2000,now U.S. Pat. No. 6,699,661.

BACKGROUND OF THE INVENTION

a) Field of the Invention

This invention relates to a method for determining a concentration of atarget nucleic acid, a nucleic acid probe for the process, and a methodfor analyzing data obtained by the method. Specifically, the presentinvention is concerned with various methods for determiningconcentrations of diverse nucleic acids on the basis of a principle thatfluorescence emission from of a fluorescent dye decreases when a nucleicacid probe labeled with the fluorescent dye is hybridized to a targetnucleic acid, that is, by measuring decreases in fluorescence emissionsfrom fluorescent dyes after hybridization of nucleic acid probes labeledwith the fluorescent dyes to target nucleic acids relative tofluorescence emissions from the fluorescent dyes before thehybridization, nucleic acid probes and devices useful for the practiceof the methods, methods for analyzing data obtained by PCR which is oneof the determination methods, systems provided with means for practicingthe analysis methods, and computer-readable recording media withindividual procedures of the analysis methods stored therein asprograms.

b) Description of the Related Art

A variety of methods are conventionally known to determine aconcentration of a nucleic acid by using a nucleic acid probe labeledwith a fluorescent-dye. These methods include:

(1) Dot Blotting Assay

After a target nucleic acid and a nucleic acid probe labeled with afluorescent dye are hybridized on a membrane, unreacted nucleic probe iswashed off. The intensity of fluorescence only from fluorescent dyemolecules, by which the nucleic acid probe hybridized with the targetnucleic acid is labeled, is measured.

(2) Method Making Use of an Intercalator: Glazer et al., Nature, 359,959, 1992

A certain specific fluorescent dye called “intercalator” emits strongfluorescence upon its insertion into a double strand of a nucleic acid.This method measures an increase in fluorescence from the fluorescentdye. Examples of the fluorescent dye can include ethidium bromide[Jikken Igaku (Laboratory Medicine), 15(7), 46-51, Yodosha (1997)] andSYBR R Green I (LightCycler™ System, Apr. 5, 1999; pamphlet distributedby Roche Diagnostics, Mannheim, Germany).

(3) Method Making Use of FRET (Fluorescence Energy Transfer): Mergny etal., Nucleic Acid Res., 22, 920-928, 1994

This method comprises hybridizing two nucleic acid probes to a targetnucleic acid. These two nucleic acid probes are labeled by differentfluorescent dyes, respectively. The fluorescent dye of one of the twoprobes can transfer energy to the fluorescent dye of the other probesuch that the latter fluorescent dye is caused to emit fluorescence.These two probes are designed such that they hybridize with theirfluorescent dyes being located opposite each other and apart from eachother by 1 to 9 bases. When these two nucleic acid probes hybridize tothe target nucleic acid, emission of fluorescence from the latterfluorescent dye takes place. The intensity of this fluorescence emissionis proportional to the number of replications of the target nucleicacid.

(4) Molecular Beacon Method: Tyagi et al., Nature Biotech., 14, 303-308,1996

A nucleic acid probe for use in this method is labeled at an end thereofwith a reporter dye and at an opposite end thereof with a quencher dye.As both end portions of the probe are complementary with each other intheir base sequences, the overall base sequence of the probe is designedto form a hairpin stem. Owing to this structure, emission from thereporter dye is suppressed by the quencher dye under Forster resonantenergy in a state suspended in a liquid. When the probe hybridizes to atarget nucleic acid, the hairpin stem structure is broken. This leads toan increase in the distance between the reporter pigment and thequencher pigment, so that the transfer of Forster resonant energy nolonger takes place. This allows the reporter dye to make emission.

(5) Davis's Method: Davis et al., Nucleic Acids Res., 24, 702-706, 1996

This method uses DNA constructs containing one or two fluoresceinmolecules in flow cytometry. The fluorescein molecules were attached tothe 3′ end of a DNA probe through an 18-atom spacer arm that resulted ina 10-fold increase in fluorescence intensity compared to the DNA probeto which fluorescein was directly attached to the 3′ end of the probe.

Applied to various determination methods for nucleic acids, fluorescentin situ hybridization assays, PCR, lighase chain reactions, stranddisplacement assays, competitive hybridization and the like, significantdevelopments have been made on these methods.

Although these methods are now widely used, they include adisadvantageous step that, subsequent to hybridization reaction betweena nucleic acid probe labeled with a fluorescent dye and a target nucleicacid, an unhybridized portion of the nucleic acid probe has to be washedout of the reaction system. Obviation of this step can apparently bringabout shorter determination time, simplified determination, and accuratedetermination. There is, accordingly, a long-standing desire for thedevelopment of a nucleic acid determination method which does notinclude such a step.

SUMMARY OF THE INVENTION

With the foregoing in view, the present invention has as an objectthereof the provision of a method for determining a concentration of atarget nucleic acid by using a nucleic acid probe labeled with afluorescent dye, which makes it possible to determine the concentrationof the target nucleic acid in a shorter time, more easily and moreaccurately.

The present invention also has as other objects the provision of nucleicacid probes and devices useful for the practice of the methods, methodsfor analyzing data obtained by PCR which is one of the determinationmethods, systems provided with means for practicing the analysismethods, and computer-readable recording media with individualprocedures of the analysis methods stored therein as programs.

To achieve the above-described objects, the present inventors haveproceeded with an investigation on methods for determining aconcentration of a nucleic acid by using a nucleic acid probe. As aresult, it was found that emission of fluorescence from a fluorescentdye decreases (quenching phenomenon of fluorescence) when a nucleic acidprobe labeled with the fluorescent dye hybridizes to a target nucleicacid.

It was also found that this decrease is significant with certainspecific dyes. It was also found that the extent of this decrease variesdepending on bases in a probe portion, to which the fluorescent dye isconjugated, or on the sequence of the bases. The present invention hasbeen completed base on these findings.

Therefore, the present invention provides the following methods, probes,kits, systems, recording medium, device and assay:

1) A method for determining a concentration of a target nucleic acid byusing a nucleic acid probe labeled with a fluorescent dye, whichcomprises:

-   -   providing, as the probe, a nucleic acid probe capable of        reducing fluorescence emission from the fluorescent dye when        hybridized with the target nucleic acid;    -   hybridizing the probe to the target nucleic acid; and    -   measuring a decrease in fluorescence emission from the        fluorescent dye after the hybridization relative to fluorescence        emission from the fluorescent dye before the hybridization.        2) A nucleic acid probe for determining a concentration of a        target nucleic acid, said probe being labeled with a fluorescent        dye, wherein:    -   the probe is labeled at an end portion thereof with the        fluorescent dye, and    -   the probe has a base sequence designed such that, when the probe        is hybridized with the target nucleic acid, at least one G        (guanine) base exists in a base sequence of the target nucleic        acid at a position 1 to 3 bases apart from an end base portion        where the probe and the target nucleic acid are hybridized with        each other,    -   whereby the fluorescent dye is reduced in fluorescence emission        when the probe is hybridized with the target nucleic acid; and    -   a method for determining a concentration of a target nucleic        acid, which comprises hybridizing the above-described nucleic        acid probe to the target nucleic acid and measuring a decrease        in fluorescence emission from the fluorescent dye after the        hybridization relative to fluorescence emission from the        fluorescent dye before the hybridization.        3) A method for analyzing or determining polymorphism or        mutation of a target nucleic acid or gene, which comprises:    -   hybridizing the above-described nucleic acid probe to the target        nucleic acid or gene, and    -   measuring a change in fluorescence.        4) A kit for analyzing or determining polymorphism or mutation        of a target nucleic acid or gene, comprising the above-described        nucleic acid probe.        5) A method for analyzing data obtained by the analysis or        determination method described above under 3), which comprises        the following step:    -   correcting a fluorescence intensity of a reaction system, in        which the target nucleic acid or gene has been hybridized with        the nucleic acid probe labeled with the fluorescent dye, in        accordance with a fluorescence intensity of the reaction system        before the hybridization.        6) A system for analyzing or determining polymorphism or        mutation of a target nucleic acid or gene, comprising means for        practicing the data analysis or determination method described        above under 5).        7) A computer-readable, recording medium comprising a program        recorded therein for making a computer perform the correction        step described above under 5).        8) A method for determining a concentration of a target nucleic        acid, which comprises using a probe with the nucleic acid probe        described above under 1) or 2) bound on a surface of a solid        support; and    -   a device for determining a concentration of the target nucleic        acid, which is useful in practicing the above method.        9) A fluorescent in situ hybridization assay making use of the        nucleic acid determination method described above under 1), 2)        or 8).        10) A method for analyzing data obtained by the nucleic acid        determination method described above under 1), 2) or 9).        11) A PCR method making use of the nucleic acid determination        method described above under 1) or 2).        12) A method for analyzing data obtained by the PCR method        described above under 11).        13) A method for analyzing a melting curve of a nucleic acid by        using the PCR method described above under 11).        14) An analysis method making combined use of the methods        described above under 12) and 13), respectively.        15) A PCR determination and/or analysis system provided with        means for performing an analysis in accordance with the analysis        method described above under 11), 12), 13) or 14).        16) A computer-readable recording medium with procedures,        through which an analysis is performed by the analysis method        described above under 11), 12), 13) or 14), recorded as a        program.        17) A method for quantitating a target nucleic acid, which        comprises making use of the data analysis method described above        under 12) or 14).        18) A method for determining a concentration of a target nucleic        acid, which comprises using the PCR determination and/or        analysis system described above under 15).        19) A method for determining a concentration of a target nucleic        acid, which comprises using the recording medium described above        under 16).

Numerous advantageous effects have been brought about by the presentinvention as will be set out below.

1) Since use of the nucleic acid determination method, probe or deviceaccording to the present invention does not require an operation such asthat needed to remove unreacted nucleic acid probe from a determinationsystem, the concentration of a target nucleic acid can be determined ina short time and with ease. When applied to a co-cultivation system ofmicroorganisms or a symbiotic cultivation system of microorganisms, theviable count of a particular microorganism strain in the system can bespecifically measured in a short time. Further, the present inventionhas also provided a simple method for analyzing or determiningpolymorphism, such as SNP (single nucleotide polymorphism), or mutationof a target nucleic acid or gene.2) Further, the quantitative PCR method according to the presentinvention has the following advantageous effects:

-   -   a. As the quantitative PCR method does not involve addition of        any factor which may act in an inhibitive manner on        amplification of a target nucleic acid by Taq DNA polymerase,        quantitative PCR can be conducted under similar conditions as        conventionally-known usual PCR having specificity.    -   b. The specificity of PCR can be maintained high, so that        amplification of primer dimer is retarded. Compared with        conventionally-known quantitative PCR, the quantitation limit        can be lowered on the order of about one digit.    -   c. It is no longer required to provide a complex nucleic acid        probe. It is, therefore, possible to save time and cost which        would otherwise be required for such a complex nucleic acid        probe.    -   d. A target nucleic acid can be effectively amplified, so that        the amplification step can be monitored in real time.        3) Upon analysis of data obtained by the real-time quantitative        PCR, the data analysis method according to the present invention        can be used to prepare a working line for the determination of        the number of copies of a nucleic acid in a nucleic acid sample        of unknown nucleic acid copy number. This working line has a        correlation coefficient which is far higher than those available        by conventional methods. Use of the data analysis method        according to the present invention, therefore, makes it possible        to accurately determine the number of copies of nucleic acid.        4) A working line the correlation efficient of which is high can        be automatically prepared by the use of the data analysis        software relating to the analysis method of data obtained by        real-time quantitative PCR, the computer-readable recording        medium with the procedures of the analysis method recorded as a        program therein, or the determination or analysis system for the        real-time quantitative PCR. The data analysis software,        computer-readable recording medium, and the determination or        analysis system all pertain to the present invention.        5) Further, use of the novel method according to the present        invention for the analysis of the melting curve of a nucleic        acid makes it possible to determine the Tm value of the nucleic        acid with high accuracy. Moreover, use of the data analysis        software for the method, the computer-readable recording medium        with the procedures of the analysis method recorded as a program        therein, or the determination or analysis system for the        real-time quantitative PCR makes it possible to obtain an        accurate Tm value.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing measurement data of fluorescence intensitywhen the sequence of bases in 16S rRNA of Escherichia coli, said basesranging from the 335^(th) base to the 358^(th) base as counted from the5′ end, was determined using a nucleic acid probe obtained in Example 1;

FIG. 2 diagrammatically illustrates effects of heat treatment of 16SrRNA on hybridization of a 35-nucleotides-chained 2-O-Me probe to atarget nucleic acid, in which:

-   -   Dashed curve: Heated 16S rRNA was added to a solution of the        probe, and    -   Solid curve: Non-heated 16S rRNA was added to a solution of the        probe;

FIG. 3 diagrammatically shows effects of methylation of 2′-OH of ribosein the 5′ terminal residue of the probe, the number of nucleotide-chainof the probe and the helper probes on the hybridization of the probes tothe target 16S rRNA;

FIG. 4 shows a working curve of rRNA assay by an invention method;

FIG. 5 diagrammatically shows analysis results of time-dependent rRNA ofthe strains, KYM-7 and KYM-8, in co-cultivation by an in situhybridization assay method with the invention technique;

FIG. 6 is a schematic illustration of a DNA chip according to thepresent invention;

FIG. 7 diagrammatically illustrates a relationship between cycles and adecrease in fluorescence emission from a fluorescent dye in a real-timequantitative PCR method making use of primers 1 and 2 labeled withBODIPY FL, in which signs {circle around (1)} to {circle around (8)}have the following meanings:

-   -   {circle around (1)} Number of copies of E. coli genome DNA: 0:        primer: primer 1+primer 2.    -   {circle around (2)} Number of copies of E. coli genome DNA:        2.4×10⁶; primer: primer 1+primer 2.    -   {circle around (3)} Number of copies of E. coli genome DNA:        2.4×10⁵; primer: primer 1+primer 2.    -   {circle around (4)} Number of copies of E. coli genome DNA:        2.4×10⁴; primer: primer 1+primer 2.    -   {circle around (5)} Number of copies of E. coli genome DNA:        2.4×10³; primer: primer 1.    -   {circle around (6)} Number of copies of E. coli genome DNA:        2.4×10²; primer: primer 1.    -   {circle around (7)} Number of copies of E. coli genome DNA:        2.4×10¹; primer: primer 1.    -   {circle around (8)} Number of copies of E. coli genome DNA:        2.4×10⁰; primer: primer 1.

FIG. 8 diagrammatically shows a relationship between cycles and thelogarithm of a decrease in fluorescence emission from a fluorescent dyein the real-time quantitative PCR making use of primers 1 and 2 labeledwith BODIPY FL, in which signs {circle around (1)} to {circle around(8)} have the same meanings as defined above in connection with FIG. 7;

FIG. 9 is a diagram showing a working line for 16S rDNA of Escherichiacoli, which was prepared using the real-time quantitative PCR accordingto the present invention;

FIG. 10 (upper diagram) depicts decreases (%) in fluorescence intensityin real-time quantitative PCR according to the present invention inwhich a single probe of the present invention was used as opposed to twoprobes required for a conventional real-time quantitative PCR methodusing FRET;

FIG. 10 (lower diagram) shows a working line for 16S rDNA of Escherichiacoli, and the working line depicts a relationship between the number ofcopies of the 16S rDNA and starting cycles (threshold cycle numbers) ineach copy of the nucleic acid at which the decrease in fluorescenceemission was capable of being significantly observed in the upperdiagram;

FIG. 11 depicts fluorescence decrease curves obtained by real-timequantitative PCR according to the present invention, which used a primerlabeled with BODIPY FL, without performing correction processingaccording to the present invention, in which:

-   -   ▪ Target nucleic acid: 10 copies; Temperature of the reaction        system upon measurement of fluorescence intensity: 72° C.    -   ● Target nucleic acid: 100 copies; Temperature of the reaction        system upon measurement of fluorescence intensity: 72° C.    -   ▴ Target nucleic acid: 1,000 copies; Temperature of the reaction        system upon measurement of fluorescence intensity: 72° C.    -   ♦ Target nucleic acid: 10,000 copies; Temperature of the        reaction system upon measurement of fluorescence intensity: 72°        C.    -   □ Target nucleic acid: 10 copies; Temperature of the reaction        system upon measurement of fluorescence intensity: 95° C.,    -   ◯ Target nucleic acid: 100 copies; Temperature of the reaction        system upon measurement of fluorescence intensity: 95° C.    -   Δ Target nucleic acid: 1,000 copies; Temperature of the reaction        system upon measurement of fluorescence intensity: 95° C.    -   ⋄ Target nucleic acid: 10,000 copies; Temperature of the        reaction system upon measurement of fluorescence intensity: 95°        C.

FIG. 12 shows fluorescence decrease curves obtained by the real-timequantitative PCR according to the present invention as in FIG. 11 exceptthat on each of the curves, each decrease (%) in fluorescence emissionwas corrected assuming that the corresponding value in the 10^(th) cyclewas 1, in which:

-   -   ▪ Target nucleic acid: 10 copies; Temperature upon measurement        of fluorescence intensity: 72° C.    -   ● Target nucleic acid: 100 copies; Temperature upon measurement        of fluorescence intensity: 72° C.    -   ▴ Target nucleic acid: 1,000 copies; Temperature upon        measurement of fluorescence intensity: 72° C.    -   ♦ Target nucleic acid: 10,000 copies; Temperature upon        measurement of fluorescence intensity: 72° C.

FIG. 13 shows curves obtained by calculating, with respect to theindividual plotted values on the respective curves in FIG. 12, the ratesof decreases (the rates of changes) in fluorescence intensity inaccordance with the formula (9) and then plotting the thus-calculated,in which:

-   -   ▪ Target nucleic acid: 10 copies.    -   ● Target nucleic acid: 100 copies.    -   ▴ Target nucleic acid: 1,000 copies.    -   ♦ Target nucleic acid: 10,000 copies.

FIG. 14 shows a working line for human genome DNAs as obtained from thedata in FIG. 13, in which:

y: Number of copies of human β-globin gene,

x: cycles (Ct), and

R²: correlation coefficient.

FIG. 15 depicts curves obtained by subjecting the measurement values inthe individual cycles in FIG. 11 to correction processing in accordancewith the formula (1) and then plotting the corrected values relative totheir corresponding cycles, in which:

-   -   ▪ Target nucleic acid: 10 copies.    -   ● Target nucleic acid: 100 copies.    -   ▴ Target nucleic acid: 1,000 copies.    -   ♦ Target nucleic acid: 10,000 copies.

FIG. 16 illustrates curves obtained by plotting values, which have beenobtained by processing the processed values of the individual cycles inFIG. 15 in accordance with the formula (3), against their correspondingcycles, in which

-   -   ▪ Target nucleic acid: 10 copies.    -   ● Target nucleic acid: 100 copies.    -   ▴ Target nucleic acid: 1,000 copies.    -   ♦ Target nucleic acid: 10,000 copies.

FIG. 17 shows curves obtained by subjecting the corrected values in theindividual cycles in FIG. 16 to correction processing in accordance withthe formula (6) and then plotting the corrected values relative to theircorresponding cycles, in which:

-   -   ▪ Target nucleic acid: 10 copies.    -   ● Target nucleic acid: 100 copies.    -   ▴ Target nucleic acid: 1,000 copies.    -   ♦ Target nucleic acid: 10,000 copies.

FIG. 18 shows working lines drawn corresponding to 0.1, 0.3, 0.5, 0.7,0.9 and 1.2 chosen at will as candidates for threshold cycle numbersfrom the respective values of log (changes in fluorescence, %) in FIG.16, in which the individual working lines have the following correlationcoefficients:

-   -   ▴ log (change in fluorescence, %)=0.1; correlation coefficient:        0.998    -   ▪ log (change in fluorescence, %)=0.3; correlation coefficient:        0.999    -   ● log (change in fluorescence, %)=0.5; correlation coefficient:        0.9993    -   Δ log (change in fluorescence, %)=0.7 correlation coefficient:        0.9985    -   □ log (change in fluorescence, %)=0.9 correlation coefficient:        0.9989    -   ◯ log (change in fluorescence, %)=1.2 correlation coefficient:        0.9988

FIG. 19 depicts fluorescence decrease curves when real-time quantitativePCR according to the present invention was conducted on human genome DNAof 1 copy and 10 copies by using the primer labeled with BODIPY FL andthe correction processing of the formula (1) was applied, in which:

1: target nucleic acid=0 copy,

2: target nucleic acid=1 copy, and

3: target nucleic acid=10 copies;

FIG. 20 illustrates melting curves of nucleic acids when a melting curveanalysis was conducted with respect to the PCR amplification productsshown in FIG. 19, in which:

1: target nucleic acid=0 copy,

2: target nucleic acid=1 copy, and

3: target nucleic acid=10 copies; and

FIG. 21 illustrates curves obtained by differentiating the curves ofFIG. 20 and showing Tm values as valleys, in which:

2: target nucleic acid: 1 copy, and

3: target nucleic acid: 10 copies.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The present invention will next be described in further detail based oncertain preferred embodiments.

The terms as used herein—such as DNAs, RNAs, cDNAs, mRNAs, rRNAs, XTPs,dXTPs, NTPs, dNTPs, nucleic acid probes, helper nucleic acid probes (ornucleic acid helper probes, or simply helper probes), to hybridize,hybridization, intercalators, primers, annealing, extending reactions,thermal denaturing reactions, nucleic acid melting curves, PCR, RT-PCR,RNA-primed PCR, stretch PCR, reverse RCR, PCR using mixed primers, PCRusing PNA, hybridization assays, fluorescent in situ hybridizationassays, polymerase chain assays (PCR methods), ligase chain reactions,strand displacement assays, competitive hybridization, DNA chips,nucleic acid detecting (gene-getecting) devices, SNP (single nucleotidepolymorphism), and co-cultivation systems of plural microorganisms—havethe same meanings as the corresponding terms generally employed thesedays in molecular biology, genetic engineering, bioengineering and thelike.

A first feature of the present invention resides in that, in a methodfor determining a concentration of a target nucleic acid by using anucleic acid probe labeled with a fluorescent dye, a decrease influorescent emission from the fluorescent dye, said decrease takingplace upon hybridization of the nucleic acid probe to the target nucleicacid, in other words, a decrease in fluorescent emission from thefluorescent dye after the hybridization relative to fluorescent emissionfrom the fluorescent dye before the hybridization is measured.

The expression “measurement of a target nucleic acid” as used hereinmeans quantitation or quantitative detection of the target nucleic acidor mere detection of the target nucleic acid.

The expression “method for determining a concentration of a targetnucleic acid by using a nucleic acid probe labeled with a fluorescentdye” means a hybridization assay, fluorescent in situ hybridizationassay, polymerase chain assay (PCR method), ligase chain reaction,strand displacement assay, competitive hybridization or the like.According to these methods, subsequent to addition of a nucleic acidprobe labeled with a fluorescent dye, the fluorescent dye in theunreacted nucleic acid probe not hybridized with a target nucleic acidis removed from the determination system by washing or the like. Thefluorescent dye, with which the nucleic acid probe hybridized with thetarget nucleic acid is labeled, is caused to emit fluorescence from theprobe directly or by applying an indirect measure to the probe (forexample, causing an enzyme to act), and the intensity of thefluorescence is measured. The present invention is characterized in thata concentration of a target nucleic acid is determined without needingsuch a complex operation.

The term “target nucleic acid” as used herein means a nucleic acid thequantitation or qualitative detection or mere detection of which isintended, irrespective whether it is in a purified form or not andfurther irrespective of its concentration. Various other nucleic acidsmay also exist together with the target nucleic acid. For example, thetarget nucleic acid may be a specific nucleic acid in a co-cultivationsystem microorganisms (a mixed system of RNAs or gene DNAs of pluralmicroorganisms) or a symbiotic cultivation system of microorganisms (amixed system of RNAs or gene DNAs of plural animals, plants and/ormicroorganisms), the quantitation or qualitative detection or meredetection of which is intended. Purification of the specific nucleicacid, if needed, can be conducted by a method known per se in the art.For example, purification can be effected using a purification kit orthe like available on the market. Specific examples of theabove-described nucleic acid can include DNAs, RNAs, PNAs, 2-O-MeRNAs,deoxyribooligonucleotides, and riboxyorigonucleotides.

In the present invention, fluorescent dyes commonly employed for thedetermination or detection of nucleic acids by labeling nucleic acidprobes may be conveniently used. It is, however, preferred to usefluorescent dyes the fluorescence emission from each of which decreaseswhen a nucleic acid probe labeled with the fluorescent dye is hybridizedto a target nucleic acid. Illustrative of such fluorescent dyes arefluorescein and derivatives thereof [for example, fluoresceinisothiocyanate (FITC) and its derivatives]; Alexa 488, Alexa 532, cy3,cy5, EDANS (5-(2′-aminoethyl)amino-1-naphthalene sulfonic acid);rhodamine 6G (R6G) and its derivatives [for example,tetramethylrhodamine (TMR), tetramethylrhodamine isothiocyanate(TMRITC), x-rhodamine, Texas red, “BODIPY FL” (trade name, product ofMolecular Probes, Inc. (Eugene, Oreg., U.S.A.), “BODIPY FL/C3” (tradename, product of Molecular Probes, Inc.), “BODIPY FL/C6” (trade name,product of Molecular Probes, Inc.), “BODIPY 5-FAM” (trade name, productof Molecular Probes, Inc.), “BODIPY TMR” (trade name, product ofMolecular Probes, Inc.), and derivatives thereof (for example, “BODIPYTR” (trade name, product of Molecular Probes, Inc.), “BODIPY R6G” (tradename, product of Molecular Probes, Inc.), “BODIPY 564” (trade name,product of Molecular Probes, Inc.), and “BODIPY 581” (trade name,product of Molecular Probes, Inc.)]. Among these, FITC, EDANS, TMR,Alexa 488, Alexa 532, “BODIPY FL/C3” and “BODIPY FL/C6” are preferred,with Alexa 488, Alexa 532, “BODIPY FL/C3” and “BODIPY FL/C6” being morepreferred.

The nucleic acid probe which is to be hybridized to the target nucleicacid may be formed of either an oligodeoxyribonucleotide or anoligoribonucleotide. The nucleic acid probe may be a chemiricoligonucleotide which contains both of them. It is also possible to usea 2′-o-methyloligoribonucleotide in which the nucleoside portion at the5′ end of the oligoribonucletide is cytidine and the 2′-OH group of thecytidine is modified by a methyl group. To enhance affinity with RNA,the 2′-o-methyloligoribonucleotide may be inserted in anoligodeoxynucleotide.

Incidentally, a nucleic acid probe with modified DNA, such as2′-o-methyloligoribonucleotide, inserted in an oligodeoxynucleotide isused primarily for the determination of RNA. Upon determination of RNAby the probe, it is preferred to subject an RNA solution as a sample toheat treatment at 80 to 100.degree.C., preferably 90 to 100° C., mostpreferably 93 to 97° C. for 1 to 15 minutes, preferably 2 to 10 minutes,most preferably 3 to 7 minutes before hybridization with the probe suchthat the higher-order structure of RNA can be degraded. Where the basestrand of the nucleic acid probe is formed of 35 or fewer bases,addition of a helper probe, for example, an oligonucleotide the basesequence of which is (5′) AGGCCGGCCCTTGACTTTCCT (3′) (SEQ ID NO. 1) to areaction mixture is preferred for raising the efficiency of thehybridization to the sequence region. In this case, the helper probe canbe in an oligodeoxyribonucleotide form or in a2′-o-methyloligoribonucleotide form. When a nucleic acid probe longerthan a 35-base strand is used, however, it is only necessary tothermally denature target RNA. When the nucleic acid probe according tothe present invention is hybridized to RNA as described above, thefluorescence intensity decreases corresponding to the concentration ofRNA in the reaction mixture, and RNA can be determined to a final RNAconcentration of about 1.50 pM.

In determination of RNA by a conventional hybridization assay making useof a nucleic acid probe, an oligodeoxyribonucleotide oroligoribonucleotide has been used as the nucleic acid probe. Because RNAitself has a higher-order solid structure, the efficiency ofhybridization between the probe and the target RNA was poor, resultingin guantitation of low accuracy. The conventional methods, therefore,are accompanied by irksomeness that a hybridization reaction isconducted after denaturing RNA and immobilizing denatured RNA on amembrane. The method according to the present invention, on the otherhand, uses a nucleic acid probe a ribose portion of which has beenmodified to have high affinity to a particular structural part of RNA,so that a hybridization reaction can be conducted at a highertemperature compared with the conventional methods. The above-mentionedadverse effects of the high-order structure of RNA can be overcome bysimply conducting thermal denaturation as pretreatment and using ahelper probe in combination. As a consequence, the efficiency ofhybridization in the method according to the present invention ispractically as high as 100%, leading to improvements in the accuracy ofquantitation. Further, the method according to the present invention isfar simpler and easier than the conventional methods.

The probe according to the present invention is formed of 5 to 50 bases,preferably 10 to 25 bases, most preferably 15 to 20 bases. A base numbergreater than 50 leads to lower permeability through a cell membrane whenemployed in a fluorescent in situ hybridization assay, thereby narrowingan applicable range of the present invention. A base number smaller than5, on the other hand, tends to induce non-specific hybridization and,therefore, results in a large determination error.

No particular limitation is imposed on the base sequence of the probeinsofar as the probe hybridizes specifically to the target nucleic acid.Preferably, however, the base sequence of the probe may be designed suchthat, when the nucleic acid probe labeled with the fluorescent dye ishybridized with the target nucleic acid,

(1) at least one G (guanine) base exists in the base sequence of thetarget nucleic acid at a position 1 to 3 bases apart from an end baseportion where the probe and the target nucleic acid are hybridized witheach other, or

(2) base pairs in a probe-nucleic acid hybrid complex form at least oneG (guanine) and C (cytosine) pair at the end portion.

The oligonucleotide in the nucleic acid probe in the present inventioncan be produced by a conventional production process for generaloligonucleotides. It can be produced, for example, by a chemicalsynthesis process or by a microbial process which makes use of a plasmidvector, a phage vector or the like (Tetrahedron Letters, 22, 1859-1862,1981; Nucleic Acids Research, 14, 6227-6245, 1986). Further, it issuitable to Use a nucleic acid synthesizer currently available on themarket (for example, “ABI394”, trade name, manufactured by Perkin-ElmerCorp., Norwalk, Conn., U.S.A.).

To label the oligonucleotide with the fluorescent dye, desired one ofconventionally-known labeling methods can be used (Nature Biotechnology,14, 303-308, 1996; Applied and Environmental Microbiology, 63,1143-1147, 1997; Nucleic Acids Research, 24, 4532-4535, 1996). Toconjugate a fluorescent dye molecule to the 5′ end, a spacer, forexample, —(CH₂)_(n)—SH is first introduced into a phosphate group at the5′ end by a method known per se in the art. As such a spacer-introducedderivative is available on the market, a commercial product may bepurchased (Midland Certified Reagent Company). In the above-mentionedexample, n ranges from 3 to 8 with 6 being preferred. A labeledoligonucletide can be synthesized by conjugating an SH-reactivefluorescent pigment or a derivative thereof with the spacer. Thethus-synthesized oligonucleotide, which is labeled with the flurorescentdye, can be purified by reversed phase chromatography or the like toprovide a nucleic acid probe for use in the present invention.

Further, the fluorescent dye can be conjugated to the 3′ end of theoligonucleotide. In this case, a spacer, for example, —(CH₂)_(n)—NH₂ isintroduced onto an OH group on the C atom at the 3′-position of riboseor deoxyribose. As such a spacer-introduced derivative is also availableon the market like the above-described ones, a commercial product may bepurchased (Midland Certified Reagent Company). As an alternative, aphosphate group may be introduced, followed by the introduction of aspacer, for example, —(CH₂)_(n)—SH onto the OH group of the phosphategroup. In these cases, n ranges from 3 to 8, with 4 to 7 beingpreferred. A labeled oligonucletide can be synthesized by conjugating anamino- or SH-reactive fluorescent pigment or a derivative thereof withthe spacer. The thus-synthesized oligonucleotide, which is labeled withthe flurorescent dye, can be purified by reversed phase chromatographyor the like to provide a nucleic acid probe for use in the presentinvention. For the introduction of the amino group, it is convenient touse a kit reagent [for example, “Unilink Aminomodifier” (trade name,product of Clontech Laboratories, Inc., Palo Alto, Calif., U.S.A.), or“FluoReporter Kit F-6082, F-6083, F-6084 or F-10220” (trade name,product of Molecular Probes, Inc.)]. In a manner known per se in theart, molecules of the fluorescent dye can then be conjugated to theoligoribonucleotide. It is also possible to introduce molecules of thefluorescent dye into strands of the probe nucleic acid (ANALYTICALBIOCHEMISTRY, 225, 32-38, 1998).

The nucleic acid probe according to the present invention can beprepared as described above. A preferred probe form is one labeled witha fluorescent dye at the 3′ or 5′ end and containing G or C as the baseat the labeled end. If the 5′ end is labeled and the 3′ end is notlabeled, the OH group on the C atom at the 3′-position of the 3′ endribose or deoxyribose may be modified with a phosphate group or the likealthough no limitation is imposed in this respect.

Use of the nucleic probe according to the present invention is notlimited to the determination of a nucleic acid, but it can also besuitably applied to methods for analyzing or determining polymorphism ormutation of a target nucleic acid or gene in particular, its combineduse with a DNA chip to be described subsequently herein provides aconvenient method. Described specifically, the intensity of fluorescenceupon hybridization of the nucleic acid probe of this invention with thetarget nucleic acid or gene varies depending on whether or not a GC pairis formed. It is, therefore, possible to analyze or determinepolymorphism and/or mutation of a target nucleic acid or gene byhybridizing the nucleic acid probe according to the present invention tothe target nucleic acid or gene and then measuring the intensity ofemission. Specific methods will be described in Examples. In this case,the target nucleic acid or gene can be an amplified or extracted productobtained by desired one of nucleic acid or gene amplification orextraction methods. Further, no particular limitation is imposed on thekind of the target nucleic acid. Examples of the target nucleic acid towhich the present invention is applicable can include RNAs, DNAs, PNAs,and artificially-modified nucleic acids. They are however required tocontain a guanine base in strands thereof or at ends thereof, becausethe intensity of fluorescence would otherwise not decrease. The methodof the present invention can, therefore, analyze or determine a mutationor substitution such as G→A, G←A, C→T, C←T, G→C or G←C, specifically,polymorphism such as single nucleotide polymorphism (SNP). Incidentally,it is the current practice to perform an analysis of polymorphism bydetermining the base sequence of a nucleic acid or gene in accordancewith the Maxam-Gilbert method or the dideoxy method.

Inclusion of the nucleic acid probe according to the present inventionin a kit for analyzing or determining polymorphism and/or mutation of atarget nucleic acid or gene, therefore, makes it possible to suitablyuse the kit as a kit for the analysis or determination of thepolymorphism and/or mutation of the target nucleic acid or gene.

When analyzing data obtained by the method of the present invention forthe analysis or determination of polymorphism and/or mutation of atarget gene, a processing step may be added to correct the intensity offluorescence, which is emitted from the reaction system when the targetnucleic acid has hybridized to the nucleic acid probe labeled with thefluorescent dye, by the intensity of fluorescence emitted from thereaction system when the target nucleic acid and the nucleic acid probehave not hybridized with each other. The data so processed are providedwith high reliability. Accordingly, the present invention also providesa data analysis method for the method which analyzes or measurespolymorphism and/or mutation of the target nucleic acid or gene.

The present invention also features a system for analyzing ordetermining polymorphism and/or mutation of a target nucleic acid orgene, which processing means for correcting a fluorescence intensity ofa reaction system, in which the target nucleic acid or gene hashybridized to the nucleic acid probe labeled with the fluorescent dye,in accordance with a fluorescence intensity of the reaction system inwhich the target nucleic acid or gene has not hybridized to the nucleicacid probe labeled with the fluorescent dye.

The present invention further features a computer-readable recordingmedium with a program recorded therein for making a computer perform aprocessing step in which, when analyzing data obtained by the method foranalyzing or determining polymorphism and/or mutation of a targetnucleic acid or gene, a fluorescence intensity of a reaction system, inwhich the target nucleic acid or gene has hybridized to the nucleic acidprobe labeled with the fluorescent dye, is corrected in accordance witha fluorescence intensity of the reaction system in which the targetnucleic acid or gene has not hybridized to the nucleic acid probelabeled with the fluorescent dye.

The probe according to the present invention may be immobilized on asurface of a solid (support layer), for example, on a surface of a slideglass. In this case, the probe may preferably be immobilized on the endnot labeled with the fluorescent dye. The probe of this form is nowcalled a “DNA chip”. These DNA chips can be used for monitoring geneexpressions, determining base sequences, analyzing mutations oranalyzing polymorphisms such as single nucleotide polymorphism (SNP).Needless to day, they can also be used as devices (chips) fordetermining nucleic acids.

To bind the probe of the present invention, for example, to a surface ofa slide glass, a slide glass coated with polycations such as polylysine,polyethyleneimine or polyalkylamine, a slide glass with aldehyde groupsintroduced thereon, or a slide glass with amino groups introducedthereon is first provided. Binding can then be achieved, for example, byi) reacting phosphate groups of the probe to the slide glass coated withthe polycations, ii) reacting a probe, in which amino groups have beenintroduced, to the slide glass on which aldehyde groups have beenintroduced or iii) reacting a probe, in which PDC (pyridiniumdichlomate) residual groups, amino groups or aldehyde groups have beenintroduced, to the slide glass on which amino groups have beenintroduced (Fodor, P. A., et al., Science, 251, 767-773, 1991; Schena,W., et al., Proc. Natl. Acad. Sci., U.S.A., 93, 10614-10619, 1996;McGal, G., et al., Proc. Natl. Acad. Sci., U.S.A., 93, 13555-13560,1996; Blanchad, A. P., et al., Biosens. Bioelectron., 11, 687-690, 1996)

A device having nucleic acid probes arranged and bound in an array formon a surface of a solid support permits more convenient determination ofa nucleic acid.

In this case, formation of a device by individually binding many probesof this invention, the base sequences of which are different, on asurface of the same solid support makes it possible to simultaneouslydetect and quantitate a variety of genes.

Preferably, this device may be designed such that each probe is providedon a side of the solid support, said side being opposite to the side towhich the probe is bound, with at least one temperature sensor and atleast one heater and an area of the solid support, where the probe isbound, can be controlled to meet optimal temperature conditions.

For this device, probes other than those of the present invention, forexample, nucleic acid probes of a construction designed such that twodifferent fluorescent dyes are contained per molecule and each of theprobes either quenches or emits fluorescence owing to interactionbetween the two fluorescent dyes when the probe is not hybridized withits corresponding target nucleic acid but either emits fluorescence orquenches when the probe hybridizes to the target nucleic acid,specifically, a device with molecular beacons described above (Tyagi etal., Nature Biotech., 14, 303-308, 1996) or the like bound thereon canalso be used suitably. These devices, therefore, are embraced within thetechnical scope of the present invention.

Fundamental operations in the determination method making use of thedevice according to the present invention are simply to place asolution, which contains a target nucleic acid such as mRNA, cDNA orrRNA, on the solid support support on which the nucleic probes are boundand then to induce hybridization. As a result, a change in the intensityof fluorescence takes place corresponding to the concentration of thetarget nucleic acid, and the target nucleic acid can then be detectedand quantitated from the change in the intensity of fluorescence.Further, binding of many nucleic acid probes of different base sequenceson a surface of a single support makes it possible to detect andquantitate many nucleic acids at the same time. As this device can beused for exactly the same application as a DNA chip, it is a novel DNAchip. Under reaction conditions optimal for the target nucleic acid, theintensities of fluorescence emitted from the nucleic acids other thanthe target nucleic acid remain unchanged. No operation is, therefore,needed for washing off the unreacted nucleic acids. Further, independenttemperature control of the individual nucleic acid probes by theircorresponding microheaters makes it possible to control the probes undertheir optimal reaction conditions, respectively. Accurate quantitationis therefore feasible. In addition, a dissociation curve between eachnucleic acid probe and its corresponding target nucleic acid can beanalyzed by continuously changing the temperature with the microheaterand measuring the intensity of fluorescence during the changing of thetemperature. From differences in such dissociation curves, it ispossible to determine properties of the hybridized nucleic acid and alsoto detect SNP.

According to each conventional device for determining a concentrationsof a target nucleic acid, a nucleic acid probe not modified with afluorescent dye is bound or fixed on a surface of a solid support and,subsequent to hybridization with the target nucleic acid labeled withthe fluorescent dye, an unhybridized portion of the target nucleic acidis washed off, followed by the measurement of the intensity offluorescence from the remaining fluorescent dye.

To label the target nucleic acid with the fluorescent dye, the followingsteps can be followed, for example, when specific mRNA is chosen as atarget: (1) mRNA extracted from cells is extracted in its entirety, and(2) using a reverse transcriptase, cDNA is synthesized while inserting anucleoside modified by the fluorescent dye. These operations are notneeded in the present invention.

A number of various probes are applied in spots on the device. Optimalhybridization conditions, for example, temperatures or the like fornucleic acids to be hybridized to the individual probes are differentfrom each other. Theoretically speaking, it is therefore necessary toconduct a hybridization reaction and a washing operation under optimalconditions for each probe (at each spot). This is however physicallyimpossible. For all the probes, hybridization is conducted at the sametemperature and further, washing is also carried out at the sametemperature with the same washing solution. The device is, therefore,accompanied by a drawback that a nucleic acid does not hybridizealthough its hybridization is desired or that, even if its hybridizationtakes place, the nucleic acid is readily washed off as the hybridizationis not strong. For these reasons, the accuracy of quantitation of thenucleic acid is low. The present invention does not have such a drawbackbecause the above-mentioned washing operation is not needed. Further, ahybridization reaction can be conducted at an optimal temperature foreach probe by independently arranging a microheater at the bottom ofeach spot and controlling the hybridization temperature. Accordingly,the accuracy of quantitation has been significantly improved in thepresent invention.

In the present invention, use of the above-described nucleic acid probeor device makes it possible to specifically determine a concentration ofa target nucleic acid with ease in a short time.

A description will hereinafter be made of the determination method.

In the determination method according to the present invention, theabove-described nucleic acid probe is added to a measurement system andis caused to hybridize to a target nucleic acid. This hybridization canbe effected by a conventionally-known method (Analytical Biochemistry,183, 231-244, 1969; Nature Biotechnology, 14, 303-308, 1996; Applied andEnvironmental Microbiology, 63, 1143-1147, 1997). As conditions forhybridization, the salt concentration may range from 0 to 2 molarconcentration, preferably from 0.1 to 1.0 molar concentration, and thepH may range from 6 to 8, preferably from 6.5 to 7.5.

The reaction temperature may preferably be in a range of the Tm value ofthe hybrid complex, which is to be formed by hybridization of thenucleic acid probe to the specific site of the target nucleic acid, ±10°C. This temperature range can prevent non-specific hybridization. Areaction temperature lower than Tm-10° C. allows non-specifichybridization, while a reaction temperature higher than Tm+10° C. allowsno hybridization. Incidentally, a Tm value can be determined in asimilar manner as in an experiment which is needed to design the nucleicacid probe for use in the present invention. Described specifically, anoligonucleotide which is to be hybridized with the nucleic acid probeand has a complementary base sequence to the nucleic acid probe ischemically synthesized by the above-described nucleic acid synthesizeror the like, and the Tm value of a hybrid complex between theoligonucleotide and the nucleic acid probe is then measured by aconventional method.

The reaction time may range from 1 second to 180 minutes, preferablyfrom 5 seconds to 90 minutes. If the reaction time is shorter than 1second, a substantial portion of the nucleic acid probe according to thepresent invention remains unreacted in the hybridization. On the otherhand, no particular advantage can be brought about even if the reactiontime is set excessively long. The reaction time varies considerablydepending on the kind of the nucleic acid, namely, the length or basesequence of the nucleic acid.

In the present invention, the nucleic acid probe is hybridized to thetarget nucleic acid as described above. The intensity of fluorescenceemitted from the fluorescent dye is measured both before and after thehybridization, and a decrease in fluorescence intensity after thehybridization is then calculated. As the decrease is proportional to theconcentration of the target nucleic acid, the concentration of thetarget nucleic acid can be determined.

The concentration of the target nucleic acid in the reaction mixture mayrange from 0.1 to 10.0 nM, while the concentration of the probe in thereaction mixture may range from 1.0 to 25.0 nM. Upon preparation of aworking curve, the probe may desirably be used at ratios of from 1.0 to2.5 relative to the target nucleic acid.

Upon actually determining the concentration of a target nucleic acid,the concentration of which is unknown, in a sample, a working curve isfirst prepared under the below-described conditions. A correspondingprobe is added at plural concentrations to aliquots of the sample,respectively, followed by the measurement of decreases in fluorescenceintensity. The probe concentration, which corresponds to the greatestone of the decreases in fluorescence intensity so measured, is chosen asa preferred probe concentration. Based on the decrease in fluorescenceintensity measured at the preferred probe concentration, a quantitatedvalue of the target nucleic acid can be determined from the workingcurve.

A description has been made about the principle of the method of thepresent invention for the determination of a concentration of a nucleicacid. The present invention can be applied to various nucleic aciddetermination methods, for example, fluorescent in situ hybridizationassays, PCR methods, ligase chain reactions, strand displacement assays,competitive hybridizations, and TAS methods led by the NASBA method.

Examples of these applications will hereinafter be described.

a) Application to Fluorescent In Situ Hybridization Assays

The present invention can be suitably applied to the determination ofthe concentration of a nucleic acid in cells of a cultivation system ofmicroorganisms (e.g., a co-cultivation system of microorganisms or asymbiotic cultivation system of microorganisms), in which various kindsof microorganisms are contained together or a microorganism and otheranimal- or plant-derived cells are contained together and cannot beisolated from each other, or in a homogenate or the like of the cells ofthe cultivation system. The term “microorganisms” as used herein meansmicroorganisms in general sense, and no particular limitation is imposedthereon. Examples of such microorganisms can include eukaryoticmicroorganisms and prokaryotic microorganisms, and also mycoplasmas,virus and rickettsias, The term “a nucleic acid having a specificsequence” means a nucleic acid with a base sequence specific to cells ofa cell strain which is desired to be investigated, for example, as tohow it is acting in such a microorganism. Illustrative examples caninclude 5S rRNAs, 16S rRNAs and 23S rRNAs of certain specific cellstrains and particular sequences of their gene DNAs.

According to the present invention, a nucleic acid probe is added to aco-cultivation system of microorganisms or a symbiotic cultivationsystem of microorganisms and the concentration of 5S rRNA, 16S rRNA or23S rRNA of a parituclar cell strain or its gene DNA, thereby making itpossible to determine the viable count of the particular strain in thesystem. Incidentally, a viable count of a particular cell strain in aco-cultivation system of microorganisms or a symbiotic cultivationsystem of microorganisms can be determine by adding the nucleic acidprobe to a homogenate of the system and then measuring a decrease influorescence emission from the fluorescent dye before hybridizationrelative to fluorescence emission from the fluorescent dye after thehybridization. It is to be noted that this method also falls within thetechnical scope of the present invention.

The above-described determination method can be carried out as will bedescribed hereinafter. Before the addition of the nucleic acid probe,the temperature, salt concentration and pH of the co-cultivation systemof microorganisms or the symbiotic cultivation system of microorganismsare adjusted to meet the conditions described above. It is alsopreferable to adjust the concentration of the specific cell strain,which is contained in the co-cultivation system of microorganisms or thesymbiotic cultivation system of microorganisms, to 10⁷ to 10¹⁰ cells/mL,preferably 10⁹ to 10¹⁰ cells/mL in terms of viable count. Theseadjustments can be achieved by dilution, centrifugal or likeconcentration, or the like. A viable count smaller than 10⁷ cells/mLresults in low fluorescence intensity and greater determination error. Aviable count greater than 10¹² cells/mL, on the other hand, leads toexcessively high fluorescence intensity, so that the viable count of theparticular microorganism cannot be determined quantitatively.

The concentration of the nucleic acid probe to be added depends upon theviable count of the particular cell strain in the co-cultivation systemof microorganisms or the symbiotic cultivation system of microorganismsand, at a viable count of 10⁸ cells/mL, may be in a range of from 0.1 to10.0 nM, preferably in a range of from 0.5 to 5 nM, more preferably 1.0nM. A probe concentration lower than 0.1 nM cannot provide any datawhich accurately reflects the viable count of the particularmicroorganism. The probe concentration, however, cannot be specified inany wholesale manner because it depends upon the concentration of atarget nucleic acid in cells.

Upon hybridizing the nucleic acid probe to the 5S rRNA, 16S rRNA or 23SrRNA of the particular cell strain or its gene DNA in the presentinvention, the reaction temperature may be set as described above.Further, the hybridization time may also be set as described above.

The nucleic acid probe is hybridized to the 5S rRNA, 16S rRNA or 23SrRNA of the particular cell strain or its gene DNA under such conditionsas described above. A decrease in fluorescence emission from thefluorescent dye in the co-cultivation system of microorganisms or thesymbiotic cultivation system of microorganisms before the hybridizationrelative to the corresponding fluorescence emission before thehybridization is then determined.

The decrease in fluorescence emission from the florescent dye, which isdetermined as described above, is proportional to the viable count ofthe particular cell strain in the co-cultivation system ofmicroorganisms or the symbiotic cultivation system of microorganisms,because the concentration of the 5S rRNA, 16S rRNA or 23S rRNA or itsgene DNA and the viable count of the particular cell strain areproportional to each other.

In the present invention, no particular limitation is imposed oncomponents other than the microorganisms in the co-cultivation system ofmicroorganisms or the symbiotic cultivation system of microorganisms,insofar as the components do not interfere with the hybridizationbetween the nucleic acid probe according to the present invention andthe 5S rRNA, 16S rRNA or 23S rRNA or its gene DNA and further, do notinhibit emission of fluorescence from the fluorescent dye in the nucleicacid probe. For example, phosphates such as KH₂PO₄, K₂HPO₄, NaH₂PO₄ andNa_(s)HPO₄, inorganic nitrogen compounds such as ammonium sulfate,ammonium nitrate and urea, various salts of metal ions such asmagnesium, sodium, potassium and calcium ions, various salts such as thesulfates, hydrochlorides, carbonates and the like of trace metal ionssuch as magnesium, zinc, iron and cobalt ions, and vitamins may becontained to adequate extent. If the above-described interference orinhibition is observed, it may be necessary to separate cells of theplural microorganisms from the cultivation system by an operation suchas centrifugal separation and then to resuspend them in a buffer or thelike.

Usable examples of the buffer can include various buffers such asphosphate buffer, carbonate buffer, Tris-HCl buffer, Tris-glycinebuffer, citrate buffer, and Good's buffer. The buffer should be adjustedto a concentration not inhibiting the hybridization, the FRET phenomenonor the emission of fluorescence from the fluorescent dye. Thisconcentration depends upon the kind of the buffer. The pH of the buffermay range from 4 to 12, with 5 to 9 being preferred.

b) Application to PCR Methods

The present invention can be applied to any method insofar as it is aPCR method. A description will hereinafter be made of an application ofthe present invention to a real-time quantitative PCR method.

In the real-time quantitative PCR method, PCR is conducted using aspecific nucleic acid probe according to the present invention, and adecrease in fluorescence emission from the florescent dye before areaction relative to fluorescence emission from the fluorescent dyeafter the reaction is determined in real time.

The term “PCR” as used herein means a variety of PCR methods. Examplescan include RT-PCR, RNA-primed PCR, stretch PCR, reverse PCR, PCR makinguse of an Alu sequence, multiple PCR, PCR making use of a mixed primer,and PCR making use of PNA. Further, the term “quantitative” means, inaddition to quantitation in general sense, quantitation of such anextent as detection.

As described above, the term “target nucleic acid” as used herein meansa nucleic acid the quantitation or qualitative detection or meredetection of which is intended, irrespective whether it is in a purifiedform or not and further irrespective of its concentration. Various othernucleic acids may also exist together with the target nucleic acid. Forexample, the target nucleic acid may be a specific nucleic acid in aco-cultivation system microorganisms (a mixed system of RNAs or geneDNAs of plural microorganisms) or a symbiotic cultivation system ofmicroorganisms (a mixed system of RNAs or gene DNAs of plural animals,plants and/or microorganisms), the amplification of which is intended.Purification of the target nucleic acid, if needed, can be conducted bya method known per se in the art. For example, purification can beeffected using a purification kit or the like available on the market.

The conventionally-known quantitative PCR methods individually amplify,in the presence of Mg ions, a target nucleic acid by using dATP, dGTP,dCTP, dTTP or dUTP, a target nucleic acid (DNA or RNA), Taq polymerase,a primer, and a nucleic acid labeled with a fluorescent dye or anintercalator while repeatedly changing the temperature between low andhigh levels, and monitor increases in fluorescence emission from thefluorescent dye in real time in the course of the amplification [JikkenIgaku (Laboratory Medicine), 15(7), 46-51, Yodosha (1997)].

On the other hand, the quantitative PCR method according to the presentinvention is characterized in that the target nucleic acid is amplifiedby using the nucleic probe of the present invention and a decrease influorescence emission from the fluorescent dye is determined. The numberof bases in a preferred probe of the present invention for use in thequantitative PCR according to the present invention may be from 5 to 50,preferably from 10 to 25, notably from 15 to 20. No particularlimitation is imposed on the probe insofar as it hybridizes toamplification products of the target nucleic acid in PCR cycles. Theprobe may be designed in either a forward type or a reverse type.

The followings can be mentioned as illustrative examples:

(1) A probe labeled, at a 5′ end portion, preferably, the 5′ endthereof, with a fluorescent dye useful in the practice of the presentinvention. The base sequence of the probe is designed such that, whenhybridized at the end portion with a target nucleic acid, at least one G(guanine) base exists in the base sequence of the target nucleic acid ata position 1 to 3 bases apart toward the 5′ side from the base on the 5′end at which the probe and the target nucleic acid are hybridized witheach other.

(2) A probe similar to the probe (1) except that the 3′ end is labeledwith the fluorescent dye.

(3) A probe similar to the probe (1) except that the 3′ end, forexample, the OH group on the C atom at the 3′-position of ribose ordeoxyribose at the 3′ end has been modified by a phosphate group or thelike or that the OH group at the 2′-position of ribose at the 3′ end hasbeen modified by a phosphate group or the like.

(4) A probe labeled at the 3′ end portion, preferably the 3′ end withthe fluorescent dye useful in the practice of the present invention andcontaining G or C as a base at the 3′ end.

(5) A probe similar to the probe (1) except that the 5′ end portion,preferably, the 5′ end is labeled with the fluorescent dye useful in thepractice of the present invention.

(6) A probe labeled at the 5′ end portion, preferably the 5′ end withthe fluorescent dye useful in the practice of the present invention andcontaining G or C as a base at the 5′ end.

In the case of each of the probes (4), (6), the 5′ end may not bedesigned to G or C due to the base sequence of a target nucleic acid. Ifthis should be the case, 5′-guanylic acid or 5′-cytidylic acid may beadded to the 5′ end of an oligonucleotide designed as a primer from thebase sequence of the target nucleic acid. The probe so obtained canstill achieve the objects of the present invention adequately. Theexpression “nucleic acid probe designed such that the 3′ end or 5′ endbase thereof becomes G or C” as used herein is, therefore, defined toembrace not only probes designed based on the base sequence, of thetarget nucleic acid but also probes added at the 3′ end or 5′ endthereof with 5′-guanyulic acid or 5′-cytidylic acid.

In particular, the above-described probe (1), (2), (3) or (4) isdesigned such that it is not used as a primer. PCR is conducted by usinga single probe of the present invention as opposed to two(fluorescent-dye-labeled) probes needed in a real-time quantitative PCRmethod making use of the FRET phenomenon. The probe is added to a PCRreaction system, and PCR is then conducted. During a nucleic acidextending reaction, the probe which has been in a form hybridized withthe target nucleic acid or amplified target nucleic acid is degraded bypolymerase and is dissociated off from the hybrid complex. The intensityof fluorescence of the reaction system at this time or the reactionsystem in which a nucleic acid denaturing reaction has completed ismeasured. Further, the intensity of fluorescence of the reaction systemin which the target nucleic acid or amplified target nucleic acid hashybridized with the probe (i.e., the reaction system at the time of anannealing reaction or at the time of the nucleic acid extending reactionuntil the probe is eliminated from the hybrid complex by polymerase). Bycalculating a decrease of the latter fluorescence intensity from theformer fluorescence intensity, the concentration of the amplifiednucleic acid is determined. The intensity of fluorescence is high whenthe probe has completely dissociated from the target nucleic acid oramplified target nucleic acid by the nucleic acid denaturing reaction orwhen the probe has been degraded out from the hybrid complex of theprobe and the target nucleic acid or amplified nucleic acid at the timeof extension of the nucleic acid. However, the intensity of fluorescenceof the reaction system in which an annealing reaction has been completedand the probe has fully hybridized to the target nucleic acid oramplified target nucleic acid or of the reaction system until the probeis degraded out of the hybrid complex of the probe and the targetnucleic acid or amplified target nucleic acid by polymerase at the timeof a nucleic acid extending reaction is lower than the former. Thedecrease in the intensity of fluorescence is proportional to theconcentration of the amplified nucleic acid.

In this case, each of the base sequences of the probes (2), (3), (4) maydesirably be designed such that the Tm of a hybrid complex, which isavailable upon hybridization of the probe with the target nucleic acid,falls within a range of the Tm value of the hybrid complex of the primer±15° C., preferably ±5° C. If the Tm of the probe is lower than (the Tmvalue of the primer −5° C.), especially (the Tm value of the primer −15°C.) the probe does not hybridize so that no decrease takes place in thefluorescence emission from the fluorescent dye. If the Tm of the probeis higher than (the Tm value of the primer +5° C.), especially (the Tmvalue of the primer +15° C.), the probe also hybridizes to nucleic acidor acids other than the target nucleic acid so that the specificity ofthe probe is lost.

The probes (5), (6) are added as primers to PCR reaction systems. Exceptfor the PCR method according to the present invention, no PCR method isknown to make use of a primer labeled with a fluorescent dye. As the PCRreaction proceeds, the amplified nucleic acid is progressively labeledwith the fluorescent dye useful in the practice of the presentinvention. Accordingly, the intensity of fluorescence of the reactionsystem in which the nucleic acid denaturing reaction has completed ishigh but, in the reaction system in which the annealing reaction hascompleted or the nucleic acid extending reaction is proceeding, theintensity of fluorescence of the reaction system is lower than theformer intensity of fluorescence.

The PCR reaction can be conducted under similar conditions as inconventional PCR methods. It is, therefore, possible to conductamplification of a target nucleic acid in a reaction system theconcentration of Mg ions in which is low (1 to 2 mM). Needless to say,the present invention can also be conducted even in a reaction system inwhich Mg ions are contained at such a high concentration (2 to 4 mM) asthat employed in the conventionally-known quantitative PCR methods.

In the PCR method according to the present invention, Tm value can bedetermined by conducting the PCR of the present invention and thenanalyzing the melting curve of the nucleic acid with respect to theamplification products. This method is a novel analysis method of amelting curve of a nucleic acid. In this method, the nucleic acid probeemployed as a nucleic acid probe or primer in the PCR method of thepresent invention can be used suitably.

In this case, designing of the base sequence of the probe according tothe present invention into a sequence complementary with a regioncontaining SNP (single nucleotide polymorphism) makes it possible todetect SNP from a difference, if any, in a dissociation curve of thenucleic acid from the probe of the present invention by analyzing thedissociation curve after completion of PCR. If abase sequencecomplementary with an SNP-containing sequence is used as a sequence forthe probe of the present invention, a Tm value available from adissociation curve between the sequence of the probe and theSNP-containing sequence becomes higher than a Tm value available from adissociation curve between the sequence of the probe and the SNP-freesequence.

A second feature of the present invention resides in the method foranalyzing data obtained by the above-described real-time quantitativePCR method.

A real-time quantitative PCR method is now practiced in real time by asystem which is composed of a reactor for conducting PCR, an equipmentfor detecting fluorescence emission from a fluorescent dye, a userinterface, namely, a computer-readable recording medium with variousprocedures of a data analysis method recorded as a program (also calledsequence detection software system), and a computer for controlling themand analyzing data. Determination by the present invention is alsoconducted by such a system.

A description will first be made of an analyzer for real-timequantitative PCR. Any system can be used in the present inventioninsofar as it can monitor PCR in real time. Particularly suitableexamples can include “ABI PRISM™ 7700 Sequence Detection System (SDS7700)” (manufactured by Perkin-Elmer Applied Biosystems, Inc., U.S.A.)and “LightCycler™ System” (manufactured by Roche Diagnostics, Mannheim,Germany).

The above-described reactor is an apparatus for repeatedly conducting athermal denaturing reaction of a target nucleic acid, an annealingreaction and an extending reaction of the nucleic acid (these reactionscan be repeatedly conducted, for example, by successively changing thetemperature to 95° C., 60° C. and 72° C. The detection system comprisesa fluorescence emitting argon laser, a spectrograph and a CCD camera.Further, the computer-readable recording medium with the variousprocedures of the data analysis method recorded as the program is usedby installing it in the computer, and contains a program recordedtherein for controlling the above-described system via the computer andalso for processing and analyzing data outputted from the detectionsystem.

The data analysis program recorded in the computer-readable recordingmedium comprises the following steps: measuring the intensity offluorescence cycle by cycle, displaying each measured fluorescenceintensity as a function of cycles, namely, as a PCR amplification ploton a display of the computer, calculating a threshold cycle number (Ct)at which the intensity of fluorescence is begun to be detected, forminga working line useful in determining from Ct values the number of copiesof the nucleic acid in the sample, and printing data and plot values inthe respective steps. When PCR is exponentially proceeding, a linearrelationship is established between the logarithm of the number ofcopies of the targent nucleic acid at the time of initiation of PCR andCt. It is therefore possible to calculate the number of copies of thetarget nucleic acid at the time of initiation of PCR by forming aworking line based on known copy numbers of the target nucleic acid anddetecting the Ct of a sample which contains the target nucleic acid thenumber of copies of which is unknown.

The PCR-related invention is an invention for analyzing data obtained bysuch a real-time quantitative PCR method as described above. Itsrespective features will be described hereinafter.

A first feature resides in a processing step for correcting afluorescence intensity of a reaction system, which is measured when thenucleic acid amplified in each cycle is conjugated with the fluorescentdye or when the amplified nucleic acid hybridizes to a nucleic acidprobe labeled with the fluorescent dye in the method for analyzing dataobtained by the real-time quantitative PCR method, by a fluorescenceintensity of the reaction system as obtained when the above-describedconjugate or hybrid complex has dissociated in each cycle, namely, thefirst feature resides in a correction-processing step.

As a specific example of “the reaction system . . . when the nucleicacid amplified in each cycle is conjugated with the fluorescent dye orwhen the amplified nucleic acid hybridizes to a nucleic acid probelabeled with the fluorescent dye”, a reaction system upon conducting anucleic acid extending reaction or annealing at 40 to 85° C., preferably50 to 80° C. in each cycle of PCR can be mentioned. The actualtemperature depends upon the length of the amplified nucleic acid.

Further, “the reaction system . . . when the above-described conjugateor hybrid complex has dissociated” can be a reaction system uponconducting thermal denaturation of the nucleic acid in each cycle ofPCR, specifically at a reaction temperature of from 90 to 100° C.,preferably 94 to 96° C.

Any correction processing can be used as the correction processing inthe correction processing step insofar as it conforms with the objectsof the present invention. Specifically, correction processing includinga processing step by the following formula (1) or formula (2) can beexemplified.f _(n) =f _(hyb,a) /f _(den,n)  (1)f _(n) =f _(dsD,D) /f _(hyb,n)  (2)where

-   -   f_(n): correction-processed value in an n^(th) cycle as        calculated in accordance with the formula (1) or formula (2),    -   f_(hyb,n): intensity value of fluorescence of the reaction        system available after the amplified nucleic acid has conjugated        to the fluorescent dye or the amplified nucleic acid has        hybridized to the nucleic acid probe labeled with the        fluorescent dye in the n^(th) cycle, and    -   f_(den,n): intensity value of fluorescence of the reaction        system available after the nucleic acid-fluorescent dye        conjugate has dissociated in the n^(th) cycle.

This step includes a sub-step in which correction-processed valuesobtained by the above-described processing are displayed on a computerdisplay and/or are printed or the correction-processed values arelikewise displayed and/or printed in the form of a graph as a functionof cycles.

A second feature resides in a data analysis method, which comprises:

introducing correction-processed values, which have been calculated inaccordance with the formula (1) or formula (2) in individual cycles,into the following formula (3) or formula (4) to calculate rates orpercentages of changes in fluorescence between samples in the individualcycles:F _(n) =f _(n) /f _(a)  (3)F _(n) =f _(a) /f _(n)  (4)where

-   -   F_(n): rate or percentage of a change in fluorescence in an        n^(th) cycle as calculated in accordance with the formula (3) or        formula (4),    -   f_(n): correction-processed value calculated in the n^(th) cycle        as calculated in accordance with the formula (1) or formula (2),        and    -   f_(a): correction-processed value calculated in a given cycle        before a change in f_(n) is observed as calculated in accordance        with the formula (1) or formula (2), and in general, a        correction-processed value, for example, in one of 10^(th) to        40^(th) cycles, preferably one of 15^(th) to 30^(th) cycles,        more preferably one of 20^(th) to 30^(th) cycles is adopted; and

comparing the rates or percentages of changes in fluorescence.

This step includes a sub-step in which calculated values obtained by theabove-described processing are displayed on a computer display and/orare printed or the calculated values are likewise displayed and/orprinted in the form of a graph as a function of cycles. This sub-stepmay be applied or may not be applied to the correction-processed valuesobtained by the formula (1) or formula (2).

A third feature resides in a data analysis method, which comprises thefollowing processing steps:

1) performing processing in accordance with the following formula (5),(6) or (7) by using data of rates or percentages of changes influorescence as calculated in accordance with said formula (3) or (4):log_(b)(F_(n)), ln(F_(n))  (5)log_(b){(1−F_(n))×A}, ln{(1−F_(n))×A}  (6)log_(b){(F_(n)−1)×A}, ln{(F_(n)−1)×A}  (7)where

-   -   A,b: desired numerical values, preferably integers, more        preferably natural numbers and, when A=100, b=10, {(F_(n)−1)×A}        is expressed in terms of percentage (%), and    -   F_(n): rate or percentage of a change in fluorescence in an        n^(th) cycle as calculated in accordance with the formula (3) or        formula (4),

2) determining a cycle in which said processed value of said processingstep 1) has reached a constant value,

3) calculating a relational expression between cycle of a nucleic acidsample of a known concentration and the number of copies of said targetnucleic acid at the time of initiation of a reaction, and

4) determining the number of copies of said target nucleic acid in anunknown sample upon initiation of PCR.

Preferably, these steps are performed in the order of 1)→2)→3)→4).

Each of these steps 1) to 3) includes a sub-step in which processedvalues obtained by the corresponding processing are displayed on acomputer display and/or are printed or the processed values are likewisedisplayed and/or printed in the form of a graph as a function of cycles.The step 4) should include at least a printing sub-step as the processedvalues obtained in the step 4) have to be printed, although theprocessed values obtained in the step 4) may also displayed on acomputer display.

Incidentally, the correction-processed values obtained by the formula(1) or (2) and the calculated values obtained by the formula (3) or (4)may be or may not be displayed on a computer display and/or printed inthe form of graphs as a function of cycles, respectively. Thesedisplaying and/or printing sub-steps may, therefore, be added as needed.

The above-described data analysis method is particularly effective whendecreases in fluorescence emission from the fluorescent dye are measuredin the real-time quantitative PCR method. As a specific example, thereal-time quantitative PCR method according to the present invention canbe mentioned.

A fourth feature resides in an analysis system for real-timequantitative PCR, which comprises processing and storing means forperforming a data analysis method for the above-described real-timequantitative PCR method of the present invention.

A fifth feature resides in a computer-readable recording medium withindividual procedures of a data analysis method, which is adapted toanalyze PCR by using the analysis sytem for the real-time quantitativePCR, stored as a program therein, wherein the program is designed tomake a computer perform the individual procedures of the data analysismethod of the present invention.

A sixth feature resides in a novel method for determining a nucleicacid, which comprises using the data analysis method, determinationand/or analysis system and/or recording medium of the present inventionin the nucleic acid determination method.

A seventh feature resides in a method for analyzing data obtained by theabove-described method of the present invention for the analysis of amelting curve of a nucleic acid, namely, data obtained by the method ofthe present invention in which the Tm value of the nucleic acid isdetermined by conducting PCR.

Specifically, the seventh feature resides in an analysis method, whichcomprises the following steps: gradually heating a nucleic acid, whichhas been amplified by the PCR method of the present invention, from alow temperature until complete denaturation of the nucleic acid (forexample, from 50° C. to 95° C.; measuring an intensity of fluorescenceat short time intervals (for example, at intervals equivalent to atemperature rise of from 0.2° C. to 0.5° C.) during the heating step;displaying results of the measurement as a function of time on adisplay, namely, a melting curve of the nucleic acid; differentiatingthe melting curve to obtain differentiated values (−dF/dT, F: intensityof fluorescence, T: time); displaying the differentiated values asderivatives on the display; and determining a point of inflection fromthe derivatives. In the present invention, the intensity of fluorescenceincreases as the temperature rises. Preferable results can be obtainedin the present invention by adding to the above-described step a furtherprocessing step in which in each cycle, the intensity of fluorescence atthe time of the nucleic acid extending reaction, preferably at the timeof completion of the PCR reaction is divided by the value offluorescence intensity at the time of the thermal denaturing reaction.

A measurement and/or analysis system for the real-time quantitative PCRof the present invention, said real-time quantitative PCR including themethod of the present invention for the analysis of the melting curve ofa nucleic acid to the above-described novel method of the presentinvention for the analysis of data obtained by a PCR method, also fallswithin the technical scope of the present invention.

A still further feature of the present invention resides in acomputer-readable recording medium with the individual procedures of themethod of the present invention for the analysis of the melting curve ofa nucleic acid recorded therein as a program such that the procedurescan be performed by a computer or a computer-readable recording mediumwith the individual procedures of the method of the present inventionfor the analysis of data obtained by a PCR method recorded therein as aprogram such that the procedures can be performed by a computer, whereina program designed to make the computer perform the individualprocedures of the method of the present invention for the analysis ofthe melting curve of the nucleic acid is additionally recorded.

The above-described data analysis methods, systems and recording mediaof the present invention can be used in a variety of fields such asmedicine, forensic medicine, anthropology, paleontology, biology,genetic engineering, molecular biology, agricultural science andphytobreeding. They can be suitably applied to microorganism systemscalled “co-cultivation systems of microorganisms” or “symbioticcultivation systems of microorganisms”, in each of which various kindsof microorganisms are contained together or a microorganism and otheranimal- or plant-derived cells are contained together and cannot beisolated from each other. The term “microorganisms” as used herein meansmicroorganisms in general sense, and no particular limitation shall beimposed thereon. Illustrative are eukaryotic microorganisms, prokaryoticmicroorganisms, mycoplasmas, virus and rickettsias.

The vial count of a particular cell strain in a co-cultivation system ofmicroorganisms or a symbiotic cultivation systems of microorganisms canbe determined by determining the number of copies of the 5S rRNA, 16SrRNA or 23S rRNA of the particular cell strain or its gene DNA in thesystem by using one or more of the above-described data analysismethods, systems and recording media of the present invention, becausethe number of copies of the gene DNA of 5S rRNA, 16S rRNA or 23S rRNA isspecific to each cell strain. In the present invention, the vial countof a particular cell strain can also be determined by applying thereal-time quantitative PCR of the present invention to a homogenate of aco-cultivation system of microorganisms or a symbiotic cultivationsystems of microorganisms. It shall also be noted that this method alsofalls with the technical scope of the present invention.

The present invention will next be described more specifically based onthe following Examples and Comparative Examples.

EXAMPLE 1

Preparation of a nucleic acid probe to be hybridized to the basesequence of a nucleic acid ranging from the 335.sup.th base to358.sup.th base counted from the 5′ end in 16S rRNA of Escherichia coli,namely, preparation of a nucleic acid probe having a base sequence of(3′) CCGCTCACGCATC (5′) (SEQ ID NO. 2) was conducted as will bedescribed hereinafter.

A deoxyribooligonucleotide, which had the base sequence of (3′)CCGCTCACGCATC (5′) (SEQ ID NO. 2) and contained —(CH₂)₇—NH₂ bonded tothe OH group on the carbon atom at the 3′ position of deoxyribose at the3′ end of the deoxyribooligonucleotide, was purchased from MidlandCertified Reagent Company, U.S.A. From Molecular Probes, Inc.,“FluoroReporter Kit F-6082” (trade name) was also purchased, whichcontained not only “BODIPY FL” propionic acid succinimidyl ester butalso a reagent for conjugating the compound to the amine derivative ofthe oligonucleotide. The kit was caused to act on the above-purchasedoligonucleotide, whereby a nucleic acid probe labeled with “BODIPY FL”was synthesized for use in this Example.

Purification of Synthesized Product

The synthesized product was dried into a dry product. The dry productwas dissolved in 0.5M NaHCO₃/Na₂CO₃ buffer (pH 9.0). The solution wassubjected to gel filtration through “NAP-25 Column” (trade name, productof Pharmacia AB, Uppsala, Sweden), whereby unreacted substances wereremoved. Further, reversed phase HPLC (B gradient: 15 to 65%, 25minutes) was conducted under the below-described conditions. An elutedmain fraction was collected. The collected fraction was lyophilized,whereby a nucleic acid probe was obtained with a yield of 23% ascalculated relative to 2 mM of the starting oligonucleotide.

The above-described reversed phase chromatography was conducted underthe following conditions:

Eluting solvent A: 0.05 N TEAA 5% CH₃CN

Eluting solvent B (for gradient elution): 0.05 N TEAA

-   -   40% CH₃CN

Column: CAPCEL PAK C18 (trade name), 6×250 mm

Elution rate: 1.0 mL/min

Temperature: 40° C.

Detection: 254 nm

EXAMPLE 2

Using a 200-mL Erlenmeyer flask which contained sterilized nutrientbroth (NB) (50 mL; product of Difco; composition: NB, 0.08 g/100 mL),Escherichia coli JM109 was cultured overnight at 37° C. under shaking.To the culture, an equivalent amount of 99.7% ethanol was then added. A2-mL aliquot of the ethanol-added culture was centrifuged in a 2.0-mLEppendorf centrifuge tube, whereby cells were obtained. The cells werewashed once with 30 mM phosphate buffer (sodium salt) (100 μL; pH 7.2).The cells were suspended in the phosphate buffer (100 μL) whichcontained 130 mM NaCl. The suspension was ultrasonicated for 40 minutesunder ice cooling (output: 33 W, oscillating frequency: 20 kHz,oscillation method: 0.5-second oscillation, followed by a 0.5-secondpause), whereby a homogenate was prepared.

After the homogenate was centrifuged, the supernatant was collected andwas then transferred into a cell of a fluorimeter. The cell with thesupernatant placed therein was controlled at 36° C. A solution of theabove-described nucleic acid probe, said solution having had beencontrolled to 36° C. beforehand, was added to the supernatant to give afinal concentration of 5 nM. While controlling at 36° C., E. coli 16SrRNA and the nucleic acid probe were hybridized for 90 minutes.Intensity of fluorescence emission from the fluorescent dye was thenmeasured by the fluorimeter.

As the intensity of fluorescence emission from the fluorescent dyebefore the hybridization, a value measured by using 30 mM phosphatebuffer (sodium salt), which contained 130 mM NaCl, (pH: 7.2) instead ofthe above-described supernatant was adopted. Intensity of fluorescenceemission was measured by changing the ratio of the amount of the nucleicprobe to the amount of the supernatant (exciting light: 503 nm; measuredfluorescence color: 512 nm). The results are shown in FIG. 1. As isappreciated from FIG. 1, the intensity of fluorescence emission from thefluorescent dye decreased as the ratio of the amount of the supernatantincreased. Namely, it is understood that in the present invention, themagnitude of a decrease in fluorescence emission from a fluorescent dyebecomes greater in proportion to the amount of a target nucleic acid towhich a nucleic acid probe hybridizes.

EXAMPLE 3

Preparation of Nucleic Acid Probe

An oligonucleotide, which was to be hybridized to 23S rNA of Escherichiacoli JM109, had a base sequence of (5′) CCCACATCGTTTTGTCTGGG (3′) (SEQID NO. 3) contained —(CH₂)₇—NH₂, bonded to the OH group on the carbonatom at the 3′ position of the 5′ end nucleotide of the oligonucleotide,was purchased from Midland Certified Reagent Company, U.S.A. as inExample 1. From Molecular Probes, Inc., “FluoroReporter Kit F-6082”(trade name) was also purchased, which contained not only “BODIPY FL”propionic acid succinimidyl ester but also a reagent for conjugating thecompound to the amine derivative of the oligonucleotide. The kit wascaused to act on the above-purchased oligonucleotide, whereby a nucleicacid probe labeled with “BODIPY FL” was synthesized. The synthesizedproduct so obtained was purified as in Example 1, whereby the nucleicacid probe labeled with “BODIPY FL” was obtained with a yield of 25% ascalculated relative to 2 mM of the starting oligonucleotide.

EXAMPLE 4

With Escherichia coli JM109 cells obtained in Example 2, cells ofPsuedomonas paucimobilis (now called “Sphingomonas paucimobilis) 421Y(FERM P-5122), said cells having have been obtained using the sameculture medium and cultivation conditions as in Example 2, were mixed atthe same concentration as Escherichia coli JM109 in terms of OD660value, whereby a co-cultivation system of the microorganisms wasprepared. From the resulting mixed system in which the cellconcentration of Escherichia coli JM109 was the same as that in Example2, a homogenate was prepared in the same manner as in Example 2. Anexperiment was conducted in a similar manner as in Example 2 except thatthe nucleic acid probe prepared in Example 3 was used, 543 nm excitinglight was used, and 569 nm flourescence was measured. The results weresimilar to those obtaineed in Example 2.

EXAMPLE 5

The base selectivity of a target nucleic acid in the quenchingphenomenon fluorescence, that is, the base selectivity according to thepresent invention was investigated. Ten kinds of syntheticdooxyribooligonucleotides (30 mer; poly a to poly j), which will bedescribed subsequently herein, were prepared by a DNA synthesizer,“ABI394” (trade name; manufactured by Perkin-Elmer Corp.)

Also prepared were the below-described probes according to the presentinvention, which were labeled with “BODIPY FL” at the 5′ ends ofdeoxyribooligonucleotides corresponding to the above-described syntheticDNAs, respectively.

Primer DNAs, which corresponded to the above-described synthetic DNAsand contained —(CH₂)₆—NH₂ bonded to the phosphate groups at the 5′ endsof the primer DNAs, were purchased from Midland Certified ReagentCompany. From Molecular Probes, Inc., “FluoroReporter Kit F-6082” (tradename) was also purchased, which contained not only “BODIPY FL” propionicacid succinimidyl ester but also a reagent for conjugating the compoundto the amine derivative of the oligonucleotide. The kit was caused toact on the above-purchased primer DNAs, whereby invention nucleic acidprobes labeled with “BODIPY FL” (probes a, b, c, d, f, g, h) weresynthesized. An investigation was made under the below-describedconditions to determine how much the fluorescence emission from thefluorescent dye would decrease when the probes were caused to hybridizeto their corresponding synthetic deoxyribooligonucleotides, and thespecificity of the invention probes was studied.

Name Target deoxyribooligonucleotide (SEQ ID NOS. 4-13) poly a5′ATATATATTTTTTTTGTTTTTTTTTTTTTT3′ poly b5′ATATATATTTTTTTTTGTTTTTTTTTTTTT3′ poly c5′ATATATATTTTTTTTTTGTTTTTTTTTTTT3′ poly d5′ATATATATTTTTTTTTTTGTTTTTTTTTTT3′ poly e5′ATATATATTTTTTTTTTTTGTTTTTTTTTT3′ poly f5′ATATATATTTTTTTTCTTTTTTTTTTTTTT3′ poly g5′ATATATATTTTTTTTTCTTTTTTTTTTTTT3′ poly h5′ATATATATTTTTTTTTTCTTTTTTTTTTTT3′ poly i5′ATATATATTTTTTTTTTTCTTTTTTTTTTT3′ poly j5′ATATATATTTTTTTTTTTTCTTTTTTTTTT3′ Invention probe (SEQ ID NOS. 14-20)Probe a 3′TATATATAAAAAAAACAA5′-BODIPY FL/C6 Probe b3′TATATATAAAAAAAAACA5′-BODIPY FL/C6 Probe c3′TATATATAAAAAAAAAAC5′-BODIPY FL/C6 Probe d3′TATATATAAAAAAAAAAA5′-BODIPY FL/C6 Probe f3′TATATATAAAAAAAAGAA5′-BODIPY FL/C6 Probe g3′TATATATAAAAAAAAAGA5′-BODIPY FL/C6 Probe h3′TATATATAAAAAAAAAAG5′-BODIPY FL/C6

(1) Components of hybridization mixure Synthetic DNA 320 nM (finalconcentration) Nucleic acid probe 80 nM (final concentration) NaCl 50 nM(final concentration) MgCl₂ 1 nM (final concentration) Tris-HCl buffer(pH 7.2) 100 nM (final concentration) “MiliQ” purified water 1.6992 mLFinal whole volume 2.0000 mL (2) Hybridization temperature: 51° C. (3)Measuring conditions Exciting light: 543 nm Measured fluorescence color:569 nm

TABLE 1 Decrease in Fluorescence Nucleic acid probe Target nucleic acidintensity (%) a a −10 b b 2 c c 75 d d 48 d e 18 f f −8 g g −2 h h 70 dI −6 d j −5

The results are shown in Table 1. As is appreciated from Table 1, it ispreferred to design the base sequence of a nucleic acid probe labeledwith a fluorescent dye such that, when the nucleic acid probe ishybridized with a target DNA (deoxyribooligonucleotide), at least one G(guanine) base exists in the base sequence of the target DNA at aposition 1 to 3 bases apart from an end base portion where the probe andthe target DNA are hybridized with each other. From Table 1, it is alsounderstood to be desired to design the base sequence of a nucleic acidprobe labeled with a fluorescent dye such that, when the nucleic acidprobe is hybridized with a target, base pairs in the probe-DNA hybridcomplex form at least one G (guanine) and C (cytosine) pair at the endportion.

EXAMPLE 6

Target nucleic acids and invention nucleic acid probes of thebelow-described base sequences were prepared. In a similar manner as inthe preceding Example, an investigation was made about effects of thenumber of G(s) in each target nucleic acid and the number of G(s) in itscorresponding invention nucleic acid probe.

Name Target deoxyribooligonucleotide (SEQ ID NOS. 21-31) poly k5′TATATATATATTTTTGGGGG3′ poly l 5′TATATATATATTTTTTGGGG3′ poly m5′TATATATATTTTTTTTTGGG3′ poly n 5′TATATATATTTTTTTTTTGG3′ poly o5′TATATATATTTTTTTTTTTG3′ poly p 5′TATATATATATTTTTCCCCC3′ poly q5′TATATATATATTTTTTCCCC3′ poly r 5′TATATATATTTTTTTTTCCC3′ poly s5′TATATATATTTTTTTTTTCC3′ poly t 5′TATATATATTTTTTTTTTTC3′ poly u5′TATATATATTTTTTTTTTTT3′ Invention probe (SEQ ID NOS. 32-42) probe k3′ATATATATATAAAAACCCCC5′-BODIPY FL/C6 probe l3′ATATATATATAAAAAACCCC5′-BODIPY FL/C6 probe m3′ATATATATATAAAAAAACCC5′-BODIPY FL/C6 probe n3′ATATATATATAAAAAAAACC5′-BODIPY FL/C6 probe o3′ATATATATATAAAAAAAAAC5′-BODIPY FL/C6 probe p3′ATATATATATAAAAAGGGGG5′-BODIPY FL/C6 probe q3′ATATATATATAAAAAAGGGG5′-BODIPY FL/C6 probe r3′ATATATATATAAAAAAAGGG5′-BODIPY FL/C6 probe s3′ATATATATATAAAAAAAAGG5′-BODIPY FL/C6 probe t3′ATATATATATAAAAAAAAAG5′-BODIPY FL/C6 probe u3′ATATATATATAAAAAAAAAA5′-BODIPY FL/C6

TABLE 2 Decrease in Fluorescence Nucleic acid probe Target nucleic acidintensity (%) k k 93 l l 92 m m 94 n n 92 o o 87 p p 61 q q 68 r r 69 ss 75 t t 73 u u 2

As is appreciated from Table 2, neither the number of G(s) in a targetnucleic acid nor the number of G(s) in an invention probe substantiallyaffects a decrease in fluorescence intensity.

EXAMPLE 7

Target nucleic acids and invention nucleic acid probes of thebelow-described base sequences were prepared. In a similar manner as inthe preceding Example, an investigation was made about effects of thekind of bases in each target nucleic acid and the kind of bases in itscorresponding invention nucleic acid probe.

Name Target deoxyribooligonucleotide (SEQ ID NOS. 43-46) poly W5′CCCCCCTTTTTTTTTTTT3′ poly X 5′GGGGGGAAAAAAAAAAAA3′ poly Y5′TTTTTTCCCCCCCCCCCC3′ poly Z 5′AAAAAAGGGGGGGGGGGG3′ Invention probe(SEQ ID NOS. 47-50) probe w BODIPY FL/C6-5′AAAAAAAAAGGGGGG3′ probe xBODIPY FL/C6-5′TTTTTTTTTCCCCCC3′ probe y BODIPYFL/C6-5′GGGGGGGGGAAAAAA3′ probe z BODIPY FL/C6-5′CCCCCCCCCTTTTTT3′

TABLE 3 Fluorescence Nucleic Target Fluorescence intensity afterDecrease in acid nucleic intensity from addition of target fluorescenceprobe acid probe alone (A) nucleic acid (B) intensity, % (C)* W w 330380 −15 X x 440 430 2 Y y 40 50 25 Z z 360 30 92 *Decrease influorescence intensity, % (C) = {(A − B)/A} × 100

As is appreciated from Table 3 and the preceding Example, a substantialdecrease takes place in fluorescence intensity (i) when an end of aninvention probe labeled with a fluorescent dye is composed of C andhybridization of a target nucleic acid forms a G-C pair, or (ii) when anend of an invention probe labeled with a fluorescent dye is composed ofa base other than C and at least one G exists on a side closer to the 3′end of a target nucleic acid than a base pair formed of a base at alocation where the invention probe is labeled with the fluorescent dyeand a base of the target nucleic acid.

EXAMPLE 8

Concerning the kinds of dyes usable for labeling nucleic acid probes ofthe present invention, an investigation was made in a similar manner asin the preceding Examples. As an invention probe, the probe z of Example7 was used. As a target nucleic acid, on the other hand, theoligonucleotide z of Example 7 was employed.

The results are shown in Table 4. As is readily envisaged from thistable, illustrative fluorescent dyes suitable for use in the presentinvention can include FITC, “BODIPY FL” “BODIPY FL/C3”, 6-joe, and TMR.

TABLE 4 Fluorescent dye Decrease in fluorescence intensity (%) FITC 90“BODIPY FL” 95 “BODIPY FL/C3” 98 “BODIPY FL/C3” 97 6-joe 75 TMR 93Incidentally, the decreases (%) in fluorescence intensity werecalculated in a similar manner as in Example 7.

EXAMPLE 9

Preparation of Nucleic Acid Probe

An oligonucleotide was purchased from Midland Certified Reagent Company,U.S.A. as in Example 1. The oligonucleotide had a base sequence of

(SEQ ID. NO. 51) (5′)CATCCCCACCTTCCTCCCAGTTGACCCCGGCAGTC(3′)(35 base pairs) hybridizable specifically to the 16S rNA base sequenceof KYM-7 strain, said base sequence being equivalent to the basesequence ranging from the 1156^(th) base to the 1190^(th) base of the16S rRNA of Escherichia coli JM109, contained deoxyribonucleotides atthe 1^(st) to 16^(th) bases and the 25^(th) to 35^(th) bases,respectively, said methyl-modified ribonucleotides being modified withmethyl groups at the OH group at the 2′ position on the carbon atom orribose, and was modified with —(CH₂)₇—NH₂— at the phosphate group of the5′-terminal group of the 35 base pairs. From Molecular Probes, Inc.,“FluoroReporter Kit F-6082” (trade name) was also purchased, whichcontained not only “BODIPY FLC6” propionic acid succinimidyl ester butalso a reagent for conjugating the compound to the amine derivative ofthe oligonucleotide. The kit was caused to act on the aboveoligonucleotide, whereby a nucleic acid probe labeled with “BODIPYFL/C6” was synthesized. The synthesized product so obtained was purifiedas in Example 1, whereby the nucleic acid probe labeled with “BODIPYFL/C6” was obtained with a yield of 23% as calculated relative to 2 mMof the starting oligonucleotide. This probe was named “35-nucleotideschained 2-O-Me probe”.

Using a DNA synthesizer, a riboxyoligonucleotide having a base sequenceof (5′) AGGCCGGCCCTTGACTTTCCT (3′) (SEQ ID NO. 52) was synthesized as inthe above to provide it as a forward-type hepter probe. On the otherhand, a riboxyoligonucleotide having a base sequence of

(SEQ ID NO. 53) (5′)AUGGGAGUUCAGUAGUACCCGCAAUGCUGGUCC(3′)was synthesized by using a DNA synthesizer, thereby providing it as areverse type helper probe.

The above-described 16S rRNA was subjected to heat treatment at 95° C.for 5 minutes, and was then added to a probe solution which had beenmaintained under the below-described reaction conditions. By afluorescence measuring instrument “Perkin-Elmer LS-50B” (trade name),the intensity of fluorescence was measured. The results are shown inFIG. 2. Incidentally, data obtained by using 16S rRNA which was notsubjected to the above-described heat treatment are plotted as acontrol. It is understood from FIG. 2 that substantial decreases influorescence intensity took place in the experimental group in theexperimental group subjected to heat treament. These results indicatethat heat treatment of 16S rRNA at 95° C. induces stronger hybridizationwith the probe according to the present invention.

Reaction Conditions:

16S rRNA: 10.0 nm Probe: 25 nM Buffer: 100 mM succinic acid, 125 mMlithium hydroxide, 8.5% lithium dodecylsulfite, pH 5.1 Temperature: 70°C.

EXAMPLE 10

Effects of Helper Probe on the Efficiency of Hybridization

Invention probes and helper probes, which were to be hybridized to theabove-described 16S rRNA, were prepared in a similar manner as describedabove. Under conditions to be described subsequently herein, aninvestigation was made about effects of a 2′-O-Me probe of the presentinvention, effects of the length of a nucleotides chain in the probe andeffects of a helper probe. The results are presented in diagrams A, B, Cand D in FIG. 3. It is appreciated from these diagrams that the 2′-O-Meprobe according to the present invention contributes to the efficiencyof hybridization. It is also understood that the helper probe iseffective in increasing the efficiency of hybridization when the basestrand of the 2′-O-Me probe is short.

1) The same 35-nucleotides-chained 2′-O-Me probe as described above.

2) A probe having the same base sequence as the 35-nucleotides-chained2′-O-Me described above under i) except that the oligonucleotide isformed of a deoxyribonucleotide (33-nucleotides-chained DNA probe).

3) A probe having the same base sequence as the 35-nucleotides chained2′-O-Me described above under 1) except that the nucleotides rangingover 8 bases from the 5′ end and 16 bases from the 3′ end were removed(17-nucleotides-chained 2-O-Me probe).

4) A probe having the same base sequence as the 33-nucleotides-chained2-O-Me probe described above under 2) except that a nucleotide rangingover 16 bases from the 3′ end was removed (17-nucleotides-chained DNAprobe).

5) A helper probe obtained by modifying the OH groups of the central 8bases of the above-described forward-type helper probe with methylgroups (forward-type 2-O-Me-helper probe).

6) A helper probe obtained by modifying the OH groups of the central 8bases of the above-described reverse-type helper probe with methylgroups (reverse-type 2-O-Me-helper probe).

7) A helper probe having the same base sequence as the above-describedforward helper probe except that the oligonucleotide is formed of adeoxyribonucleotide (forward-type DNA helper probe).

8) A helper probe having the same base sequence as the above-describedreverse helper probe except that the oligonucleotide is formed of adeoxyribonucleotide (reverse-type DNA helper probe).

9) A ribooligonucleotide having a sequence of (5′)GUGACGGUCACUAUUUGACCUCCUUCCACCCC (3′) (SEQ ID NO. 54) (35-baseribooligonucleotide).

10) A ribooligonucleotide having a base sequence of (5′)GUGACGGUCACUAUUUG (3′) (SEQ ID NO. 55) (17-base ribooligoriucleotide).

Reaction Conditions:

Concentration of 16S rRNA: 10 nM Concentration of probe: 25 nM Helperprobe concentration: 1 μM Buffer composition: 100 mM succinic acid, 125mM lithium hydroxide, 8.5% lithium dodecyl-sulfite, pH 5.1

Reaction Temperature:

-   -   70° C. (for 35-nucleotides-chained 2-O-me probe)    -   65° C. (for 17-nucleotides-chained 2-O-Me probe and        33-nucleotides-chained DNA probe)    -   50° C. (for 17-nucleotides-chained DNA probe)

EXAMPLE 12

Preparation of Working Curve for rRNA Determination

At diverse concentrations within a range of from 0.1 to 10 nM, theabove-described rRNA was heated at 95° C.: for 5 minutes. The resultingnucleic acid solutions were added to aliquots of a reaction mixture,respectively. The reaction mixture had been prepared and maintainedunder the below-described reaction conditions. Upon elapsed time of1,000 seconds, decreases in fluorescence intensity were measured using“Perkin-Elmer LS-50B”. The results are plotted in FIG. 4. It isappreciated from the diagram that the working curve shows linearity inthe range of from 0.1 to 10 nM.

Reaction Conditions:

Concentrations of 33-nucleotides- 10 nM chained 2-O—Me probe: Buffercomposition: 100 mM succinic acid, 125 mM lithium hydroxide, 8.5%lithium dodecyl- sulfite, pH 5.1 Reaction temperature: 70° C.

EXAMPLE 12

Florescent In Situ Hybridization Assay

In a similar manner as described above, 35-nucleotides-chained 2-O-Meprobe and 36-nucleotides-chained 2-O-Me probe according to the presentinvention were prepared for hybridization to the respective rRNAs ofCellulomonas sp. KYM-7 (FERM P-16806) and Agrobacterium sp. KYM-8 (FERMP-11358), respectively, Those probes had the following base sequences:

35-nucleotides-chained 2-O-Me probe for assaying the rRNA ofCellulomonas sp. KYM-7:

(SEQ ID NO. 56)

(the boxed bases are modified with methyl groups)

36-nucleotides-chained 2-O-Me probe for assaying the rRNA ofAgrobacterium sp. KYM-8:

(SEQ ID NO. 57)

(the boxed bases are modified with methyl groups)

Cellulomonas sp, KYM-7 (FERM P-16806) and Agrobacterium sp. KYM-8 (FERMP-11358) were co-cultured with the below-described medium compositionunder the below-described cultivation conditions. Co-cultures weresampled at various phases of the co-cultivation. From each of theco-cultures, rRNAs were prepared using “RNeasy Maxikit” (trade name;product of QIAGEN GmbS (Hilden, Germany). Those rRNAs were heated at 95°C. for 5 minutes, and then added to the reaction mixture which had beenmaintained under the reaction conditions. After they were reacted at 70°C. for 1,000 seconds, a decrease in fluorescence intensity was measuredusing “Perkin-Elmer LS-50B”. The results are plotted in FIG. 5.Incidentally, the total rRNA was measured using “RiboGreen RNA Kit”(trade name; product of Molecular Probe, Inc. (Eugene, Oreg., U.S.A.).

As is appreciated from the diagram, the mobilizations of the rRNAs ofthe respective cell strains were consistent with that of the total rRNA.This indicates that the method of the present invention can beeffectively used in fluorescent in situ hybridization assays.

Composition of Culture Medium (g/L):

-   -   Starch, 10.0; aspartic acid, 0.1; K₂HPO₄, 5.0; KH₂PO₄, 5.0;        KH₂PO₄, 2.0; MgSO₄.7H₂O, 0.2; NaCl, 0.1; (NH₄)₂SO₄, 0.1.        Cultivation Conditions:

The above-describe cell strains were cultivated beforehand on a slantmedium. One roopful of the culture was collected from the slant medium,and was then inoculated to the above-described sterilized nutrient broth(NB) in an Erlenmeyer flask. The strains were cultured at 30° C. and 150rpm under shaking.

Reaction Conditions:

Concentrations of 33-nucleotides- 1.0 to 10 nM chained 2-O—Me probe:Buffer composition: 100 mM succinic acid, 125 mM lithium hydroxide, 8.5%lithium dodecyl- sulfite, pH 5.1 Reaction temperature: 70° C.

A method for analyzing polymorphism and mutation of a target nucleicacid or gene will hereinafter be described in Example 13.

EXAMPLE 13

Four oligonucleotides with the below-described base sequences weresynthesized using the same DNA synthesizer as that employed in Example5. Further, an invention nucleic acid probe having the below-describedbase sequence was also synthesized in a similar manner as in Example 5.The oligonucleotides were separately hybridized with the probe insolutions. An investigation was then made as to whether or not a singlebase substitution can be determined from a change in fluorescenceintensity. The base sequence of the nucleic acid probe according to thepresent invention is designed such that, if G exists at the 3′ end ofanyone of the target oligonucleotides, it matches 100% with the basesequence of the particular oligonucleotide. The hybridizationtemperature was set at 40° C. at which all base pairs between the probeand the target oligonucleotide can hybridize 100%. The concentrations ofthe probe and target oligonucleotides, the concentration of a buffersolution, a fluorimeter, fluorescence measuring conditions, experimentalprocedures, and the like were set or chosen as in Example 5.

(SEQ ID NO. 58) Invention probe: 3′TTTTTTTTGGGGGGGGC5′BODIPY FL/C6 (SEQID NO. 59) Target nucleotide No. 1: 5′AAAAAAAACCCCCCCCA3′ (SEQ ID NO.60) Target nucleotide No. 2: 5′AAAAAAAACCCCCCCCC3′ (SEQ ID NO. 61)Target nucleotide No. 3: 5′AAAAAAAACCCCCCCCCI3′ (1: hypoxanthine) (SEQID NO. 62) Target nucleotide No. 4: 5″AAAAAAAACCCCCCCCCI3′

The results are shown in Table 5. As is appreciated from the table, nochange in fluorescence intensity was observed in the case of the targetoligonucleotides Nos. 1 to 3, but in the case of the targetoligonucleotide No. 4, a decrease as much as 84% was observed.

TABLE 5 Fluorescence Target Initial fluorescence intensity afteroligonucleotide intensity (A) hybridization (B) (A − B)/B No. 1 340 350−0.03 No. 2 332 328 0.01 No. 3 343 336 0.02 No. 4 345 52 0.84

In the method of the present invention for analyzing data (for example,the data in columns A and B in Table 5) obtained by the method foranalyzing or determining polymorphism and/or mutation of a targetnucleic acid or gene (for example, the target oligonucleotide No. 1, 2,3 or 4), the processing to correct a fluorescence intensity of areaction system, said fluorescence intensity being obtained when atarget nucleic acid or gene is hybridized with a nucleic acid probelabeled with a fluorescent dye (for example, the above-described nucleicacid probe), by a fluorescence intensity of the same reaction systemwhen the target nucleic acid or gene is not hybridized with the nucleicacid probe means the calculation of (A-B)/B in Table 3.

From the above results, it has been found that, when a target nucleicacid is a double-stranded nucleic acid, substitutions of G→A, G→A, C→T,C→T, G→C and G→C can be detected.

EXAMPLE 14

A model of A DNA chip according to the present invention is illustratedin FIG. 6. Firstly, a modified probe and a surface-treated slide glassare provided first. The modified probe had been prepared by introducingan amino group onto the 3′-OH group at the 3′ end of the inventionprobe, 3′TTTTTTTTGGGGGGGGC5′ (SEQ ID NO. 63) BODIPY FL/C6, prepared inExample 13. On the other hand, the surface-treated slide glass had beenprepared by treating a slide glass with a silane coupling agent whichcontained epoxy groups as reactive groups. A solution with the modifiedprobe contained therein was applied in spots onto the surface-treatedslide glass by a DNA chip production apparatus, “GMS™ 417 ARRAYER”(manufactured by TAKARA SHUZO CO., LTD., Kyoto, Japan). As a result, themodified probe is bound at the 3′ end onto a surface of the slide glass.The slide glass is then placed for 4 hours or so in a closed vessel tobring the reaction to completion. The slide glass was alternately dippedin 0.1% SDS solution and water, twice in each of the solution and water,for about 1 minute each time. Further, the slide glass was immersed forabout 5 minutes in a boron solution, which had been prepared bydissolving NaBH₄ (1.0 g) in water (300 mL). Shortly after the slideglass was placed for 2 minutes in water of 95° C., the slide glass wasalternately dipped in 0.1% SDS solution and water, twice in each of thesolution and water, for about 1 minute each time, so that reagents werewashed off. The slide glass was then dried. As a result, a DNA chipaccording to the present invention was prepared.

Further, arrangement of a minute temperature sensor and a microheater onthe lower side of the slide glass at a position corresponding to eachspot of the modified probe makes it possible to provide the DNA chip ofthe present invention with high performance.

A description will next be made of a determination of a target nucleicacid or gene by the DNA chip. No change takes place in fluorescenceintensity where the target nucleic acid or gene is not hybridized withthe probe, where no G-C pair is formed at the fluorescent-dye-labeledend even if they are hybridized together, or where at least one G(guanine) base does not exist in the base sequence of the target nucleicacid or gene at a position 1 to 3 bases apart from an end base portionwhere the probe and the target nucleic acid or gene are hybridized witheach other. However, the intensity of fluorescence decreases whenhybridization takes place. This fluorescence intensity can be measuredby using a DNA chip analyzer, “GMS™ 418 Array Scanner” (manufactured byTAKARA SHUZO CO, LTD.).

Methods of the present invention for PCR assay will hereinafter bedescribed in Examples 15-19.

EXAMPLE 15

Using as a target nucleic acid the 16S rRNA gene in the genome DNA ofEscherichia coli, a primer labeled with “BODIPY FL/C6) was prepared forthe amplification of the nucleic acid.

Preparation of Primer 1 (Eu800R: Reverse Type)

A deoxyribooligonucleotide having a base sequence of (5′)CATCGTTTACGGCGTGGAC (3′) (SEQ ID NO. 64) was synthesized using a DNAsynthesizer, “ABI394” (trade name: manufactured by Perkin Elmer, Corp.).An oligonucleotide, which had been prepared by treating the phosphategroup at the 5′ end of the oligodeoxyribonucleotide with phosphatase toform cytosine and then bonding —(CH₂)₉—NH₂ to the OH group on the carbonatom at the 5′-position of the cytosine, was purchased from MidlandCertified Reagent Company. From Molecular Probes, Inc., “FluoroReporterKit F-6082” (trade name) was also purchased, which contained not only“BODIPY FL” propionic acid succinimidyl ester but also a reagent forconjugating the compound to the amine derivative of the oligonucleotide.The kit was caused to act on the above-purchased oligonucleotide,whereby Primer 1 of the present invention labeled with “BODIPY FL/C6”was synthesized.

Purification of Synthesized Product

The synthesized product was dried into a dry product. The dry productwas dissolved in 0.5 M Na₂CO₃/NaHCO₃ buffer (pH 9.0). The solution wassubjected to gel filtration through “NAP-25 Column” (trade name, productof Pharmacia AB, Uppsala, Sweden), whereby unreacted substances wereremoved. Further, reversed phase HPLC (B gradient: 15 to 65%, 25minutes) was conducted under the below-described conditions. An elutedmain fraction was collected. The collected fraction was lyophilized,whereby Primer 1 of the present invention was obtained with a yield of50% as calculated relative to 2 mM of the starting oligonucleotide.

The above-described reversed phase chromatography was conducted underthe following conditions:

Eluting solvent A: 0.05 N TEAA 5% CH₃CN

Eluting solvent B (for gradient elution):

-   -   0.05 N TEAA    -   40% CH₃ CN

Column: CAPCEL PAK C18 (trade name), 6.times.250 mm

Elution rate: 1.0 mL/min

Temperature: 40° C.

Detection: 254 nm

EXAMPLE 16

Preparation of Primer 2 (Eu500R/Forward: Forward Type)

Primer 2 composed of a deoxyribooligonucleotide, which had a basesequence of (5′) CCAGCAGCCGCGGTAATAC (3′) (SEQ ID NO. 65), and afluorescent dye (“BODIPY FL/C6”) labeled to the 5′ end of thedeoxyribooligonucleotide, was prepared with a yield of 50% in a similarmanner as in Example 13.

EXAMPLE 17

Using a test tube containing a liquid culture medium (5 mL; composition:NB, 0.08 g/100 mL) of sterilized nutrient broth (NB) (product of Difco),Escherichia coli JM109 was cultivated overnight at 37° C. under shaking.A 1.5-mL aliquot of the culture was centrifuged in a 1.5-mL centrifugetube, whereby cells were obtained. From the cells, genome DNA wasextracted using “DNeasy Tissue Kit” (trade name, product of QIAGENEGmbH, Hilden, Germany). The extraction was conducted following theprotocol of the kit. As a result, a 17-ng/.mu.L DNA solution wasobtained.

EXAMPLE 18

Using the genome DNA of the above E. coli strain, Primer 1 and/or Primer2, PCR reactions were conducted by a method known per se in the artwhile using “LightCycler™ System” (trade name) marketed from RocheDiagnostics, Mannheim, Germany. Operations were conducted following themanual of the system.

In the above system, PCR was conducted as specified in the manual exceptthat Primer 1 and/or Primer 2 of the present invention were used inplace of nucleic acid probes (two nucleic acid probes making use of theFRET phenomenon) and a general primer (a general primer not labed withany fluorescent dye), both of which are listed in the manual).

PCR was conducted in a hybridization mixture of the followingcomponents:

E. coli genome DNA solution 3.5 μL (final concentration: 0 to 6 ng/20μL) (final copy number: 0 to 2.4 × 10⁶ copies) Primer solution 0.8 μL(final concentration: 0.08 μM) Taq solution 10.0 μL  “MiliQ” purifiedwater 5.7 μL Final whole volume 20.0 μL 

Incidentally, the experiments were conducted by using the target nucleicacid, E. coli 16S rDNA, at the concetrations of the respectiveexperimental groups shown in the brief description of FIG. 7 and also byusing the primers in the combinations of Primer 1 and/or Primer 2 alsoshown in the brief description of FIG. 7.

The above Taq solution is a mixed solution of the following reagents:

Taq solution 96.0 μL “MiliQ” purified water 68.2 μL Taq DNA polymerasesolution 24.0 μL Taq start  3.8 μL

Incidentally, these Taq solution and Taq DNA polymerase solution wereboth included in the “DNA Master Hybridization Probe Kit” (trade name;product of Roche Diagnostics, Mannheim, Germany). Specifically, as theTag DNA polymerase solution, the 10×conc. solution (red cap) was used bydiluting it tenfold. Further, Taq start is an antibody for the Taq DNApolymerase and is marketed by Clontech Laboratories, Inc., Palo Alto,Calif., U.S.A. Addition of Taq start to a reaction mixture can suppressactivity of Taq DNA polymerase up to 70° C.

The following reaction conditions were used.

Denaturation Initial: 95° C., 120 seconds Second and onwards: 95° C.,120 seconds Annealing 57° C., 5 seconds

Measurements were conducted using “LightCycler™ System” (manufactured byRoche Diagnostics, Mannheim Germany). For each measurement, the detectorF1 was used out of the detectors F1-F3 included in the system, and thegain and excitation level of the detector were set at 10 and 75,respectively.

The results are shown in FIG. 7 and FIG. 8. It is appreciated from FIG.7 and FIG. 8 that the number of cycles at the time of observation of adecrease in fluorescence emission from the fluorescent dye and thenumber of copies of E. coli 16S rDNA as the target nucleic acid areproportional to each other. In these diagrams, decreases in fluorescenceemission from the fluorescent dye are expressed in terms of decreases inthe intensity of fluorescence.

FIG. 9 shows a working line for E. coli 16S rDNA, in which the number ofcopies of E. coli 16S rDNA is expressed as a function of cycles. Thecorrelation coefficient was 0.9973, so that extremely good correlationwas exhibited.

As is understood from the above results, use of the quantitative PCRmethod of the present invention makes it possible to count the number ofcopies of a target nucleic acid at the beginning. This means that theconcentration of the target nucleic acid can be determined.

EXAMPLE 19

In Example 18, PCR was conducted using the invention probes as primers.In this example, however, PCR according to the present invention wasconducted under the following conditions by using a primer of thepresent invention as opposed to two probes required in the conventionalmethod making use of the FRET phenomenon.

-   -   a) Target nucleic acid: 16S rDNA of Escherichia coli

b) Primers: (SEQ ID NO. 66) Forward primer E8F:(3′)AGAGTTTGATCCTGGCTCAG(5′) (SEQ ID NO. 67) Reverse primer E1492R:GGTTACCTTGTTACGACTT(5′) (SEQ ID NO. 68) c) Probe: BODIPYFL-(3′)CCTTCCCACATCGTTT(5′)

-   -   d) PCR apparatus: “LightCycler™ System” (manufactured by Roche        Diagnostics, Mannheim Germany)    -   e) Conditions for PCR:        -   Denaturation: 95° C. for 0 second            -   (95° C. for 60 seconds in the first cycle only)        -   Annealing: 50° C. for 5 seconds        -   Extension: 72° C. for 70 seconds        -   Total cycle number: 70 cycles    -   f) Fluorescence assay (measurement):    -   Assay (measurement) was performed once after each of        denaturation and annealing in each cycle.    -   g) Composition of reaction mixture:    -   Total volume: 20.mu.L    -   Amount of DNA polymerase (“TaKaRa Ex taq”): 0.5 U    -   Amount of TaqStart antibody: 0.3.mu.L    -   Concentration of primer: 0.2 M (common to both primers)    -   Concentration of probe: 0.05 μM    -   Concentration of MgCl₂: 2 mM    -   Conc. of BSA (bovine serum albumin): 0.25 mg/mL    -   Concentration of dNTPS: 0.2 mM (for each nucleotide)

The results are shown in FIG. 10. It is understood from the diagram thatthe number of cycles at the time of observation of a decrease influorescence emission from the fluorescent dye and the number of copiesof E. coli 16S rDNA as the target nucleic acid are proportional to eachother.

As is understood from the above results, use of the quantitative PCRmethod of the present invention makes it possible to count the number ofcopies of a target nucleic acid at the beginning. This means that theconcentration of the target nucleic acid can be determined.

In the subsequent Examples, the data analysis method of the presentinvention for analyzing data obtained by using the above-describedquantitative PCR method of the present invention will be described.

EXAMPLE 20

Using, as a target nucleic acid, human genome DNA (human β-globin)(TaKara Catalog Product No. 9060) (product of TAKARA SHUZO CO., LTD.,Kyoto Japan) (hereinafer called “the human genome DNA”), a primerlabeled with “BODIPY FL/C6” was prepared for the amplification of thenucleic acid.

Preparation of Primer KM38+C (Reverse Type)

A deoxyribooligonucleotide having a base sequence of (5′)CTGGTCTCCTTAAACCTGTCTTG (3′) (SEQ ID NO. 69) was synthesized using a DNAsynthesizer, “AB1394” (trade name; manufactured by Perkin Elmer, Corp.).An oligonucleotide, which had been prepared by treating the phosphategroup at the 5′ end of the oligodeoxyribonucleotide with phosphatase toform cytosine and then bonding —(CH.sub.2).sub.9 —NH.sub.2 to the OHgroup on the carbon atom at the 5′-position of the cytosine, waspurchased from Midland Certified Reagent Company. From Molecular Probes,Inc., “FluoroReporter Kit F-6082” (trade name) was also purchased, whichcontained not only “BODIPY FL” propionic acid succinimidyl ester butalso a reagent for conjugating the compound to the amine derivative ofthe oligonucleotide. The kit was caused to act on the above-purchasedoligonucleotide, whereby Primer KM38+C of the present invention labeledwith “BODIPY FL/C6” was synthesized.

Purification of Synthesized Product

The synthesized product was dried into a dry product. The dry productwas dissolved in 0.5 M Na₂ CO₃/NaHCO₃ buffer (pH 9.0). The solution wassubjected to gel filtration through “NAP-25 Column” (trade name, productof Pharmacia AB, Uppsala, Sweden), whereby unreacted substances wereremoved. Further, reversed phase HPLC (B gradient: 15 to 65%, 25minutes) was conducted under the below-described conditions. An elutedmain fraction was collected. The collected fraction was lyophilized,whereby Primer KM38+C of the present invention was obtained with a yieldof 50% as calculated relative to 2 mM of the starting oligonucleotide.

The above-described reversed phase chromatography was conducted underthe following conditions:

Eluting solvent A: 0.05 N TEAA 5% CH₃ CN

Eluting solvent B (for gradient elution):

-   -   0.05 N TEAA    -   40% CH₃ CN

Column: CAPCEL PAK C18 (trade name) 6×250 mm

Elution rate: 1.0 mL/min

Temperature: 40° C.

Detection: 254 nm

EXAMPLE 21

Preparation of Primer KM29 (Forward Type)

A deoxyribooligonucleotide having a base sequence of (5′)GGTTGGCCAATCTACTCCCAGG (3′) (SEQ ID NO. 70) was synthesized in a similarmariner as in Example 18.

COMPARATIVE EXAMPLE 1

This Comparative Example is directed to use of a data analysis softwarewhich did not include the processing step of the present invention thatan intensity of fluorescent during an extending reaction of a nucleicacid is divided using an intensity of fluorescent at the time of athermal denaturing reaction [i.e., the processing of the formula (1)].

Using the above-described human genome DNA, Primer KM38+C and PrimerKM29, PCR reactions were conducted by “LightCycler™ System”(manufactured by Roche Diagnostics, Mannheim Germany). The intensity offluorescence was measured in each cycle.

Incidentally, the PCR in this Comparative Example employed theabove-described primers labeled with the fluorescent dye, and is a novelreal-time quantitative PCR method in which a decrease in fluorescenceemission is measured rather than an increase in fluorescence emission.Analysis of data was conducted using the software of the system itself.The PCR in this Comparative Example was conducted following the manualof the system except that the invention primers KM38+C and KM29 wereused instead of the nucleic acid probes listed in the (two probes makinguse of the FRET phenomenon) or an ordinary primer (an ordinary primernot labeled with any fluorescent dye).

PCR was conducted in a hybridization mixture of the followingcomponents:

Human genome DNA 1.0 μL (final concentration: 1 to 10,000 copies) Primersolution 4.0 μL (final concentration: 0.1 μM) Taq solution 10.0 μL “MiliQ” purified water 5.0 μL Final whole volume 20.0 μL 

Incidentally, the experiments were conducted by using the human genomeat the concetrations of the respective experimental groups shown in thebrief description of FIG. 11.

The following reaction conditions were used.

Denaturation Initial: 95° C., 60 seconds Second and onwards: 95° C., 10seconds Annealing 60° C., 5 seconds DNA extending reaction: 72° C., 17seconds

Measurements were conducted using “LightCycler™ System” (manufactured byRoche Diagnostics, Mannheim Germany). For each measurement, the detectorF1 was used out of the detectors F1-F3 included in the system, and thegain and excitation level of the detector were set at 10 and 75,respectively.

PCR was conducted as described above, during which the intensities offluorescence in individual cycles were measured. The results are shownin FIG. 11. Described specifically, with respect to each of the humangenome DNAs of the respective copy numbers, the intensity offluorescence was measured at the time of a denaturing reaction and alsoat the time of a nucleic acid extending reaction, both in each cycle,and was printed. It is observed that the intensity of fluorescenceremained constant at the time of the denaturing reaction irrespective ofthe cycle but a decrease in fluorescence took place from the 25th cycleat the time of the nucleic acid extending reaction. It is alsounderstood that this decrease occurs earlier as the number of copies ofthe human genome DNA increases.

As is shown in FIG. 11, the intensities of fluorescence in initialcycles were not constant irrespective of the number of copies of thehuman genome DNA. The following steps (b)-(i) were, therefore, added tothe data analysis method for use in this Comparative Example.

(b) Assuming that the intensity of fluorescence in the 10^(th) cycle is1, the intensity of fluorescence in each cycle is converted, namely,calculation is conducted in accordance with the following formula (8):C _(n)=(72)/F ₁₀(72)  (8)where

-   -   C_(n): converted value of the intensity of fluorescence in each        cycle,    -   F_(n)(72): the intensity of fluorescence after extending        reaction at 72° C. in each cycle, and    -   F₁₀(72): the intensity of fluorescence in the 10^(th) cycle.        (c) Each converted value obtained in step (b) is displayed on a        display and/or printed as a function of cycle.        (d) From the converted value in each cycle as obtained in step        (b), the rate of a change in fluorescence intensity (decrease or        quench, %) is calculated in accordance with the following        formula (9):        F _(dn)=log {100−C _(n)×100)}  (9)        F _(dn)=2 log {1−C _(n)}  (9)        where    -   F_(dn): the rate of a change in fluorescence intensity (decrease        or quench, %), and    -   C_(n): the value obtained in accordance with the formula (8).        (e) Each converted value obtained in step (d) is displayed on a        display and/or printed as a function of cycle.        (f) Data processed in step (d) are compared with 0.5 as a        threshold, and the number of cycles the data of which reach the        threshold is counted.        (g) A graph is prepared by plotting values, which have been        counted in step (f), along X-axis and the numbers of copies        before the initiation of the reaction along Y-axis.        (h) The graph prepared in step (g) is displayed on a display or        printed.        (i) A correlation coefficient or relational formula of the line        drawn in step (h) is calculated.        (j) The correlation coefficient or relational formula calculated        in step (i) is displayed on a display or printed.

Using the above-described data analysis software, the data obtained inFIG. 11 were then processed as will be described hereinafter.

FIG. 12 is a print-out of the data processed in step (b) [process (c)].Namely, assuming that the intensity of fluorescence in the 10^(th) cyclewas 1, the fluorescence intensities in the individual cycles wereconverted, and the converted values were plotted against thecorresponding cycles.

FIG. 13 is a print-out of the data processed in step (d) [process (e)]Namely, decreases (%) (quenches, %) of the respective fluorescenceintensities were calculated from the plotted values in FIG. 12, and thevalues so calculated were plotted against the corresponding cycles.

FIG. 14 is a print-out of the graph prepared in step (g) based on thedata processed in step (f) [step (h)]. Namely, it is a graph obtained byusing a decrease of 0.5 in fluorescence intensity as a threshold,plotting along X-axis the number of cycles in which the threshold wasreached, and also plotting along Y-axis the numbers of copies of thehuman genome DNA before the initiation of the respective reactions. Thecorrelation coefficient (R2) of the line in this graph was calculated instep (i), and was then printed [step (j)]. The correlation coefficientwas 0.9514. As is understood, it was hardly possible, with thiscorrelation coefficient, to determine an accurate number of copies thiscorrelation coefficient was

EXAMPLE 22

This Example is directed to an experiment in which processing of datawas performed by using the data analysis method of the presentinvention.

PCR was conducted in a similar manner as in Comparative Example 1.

The processing of the data was performed through similar steps as inComparative Example 1 except that the following step (a) was addedbefore the step (b) and the steps (b), (d) were modified as will bedescribed below.

(a) The intensity of fluorescence in each cycle in a reaction system inwhich an amplified nucleic acid hybridized to a nucleic acid primerlabeled with a fluorescent dye [namely, the intensity of fluorescence atthe time of a nucleic acid extending reaction (72° C.)] was corrected ina correction processing step such that the intensity of fluorescence wasdivided by the intensity of fluorescence in the reaction system measuredat the time of dissociation of the hybrid complex of the amplifiednucleic acid and the nucleic acid primer [namely, the intensity offluorescence at the time of the nucleic acid denaturing reaction (95°C.)], that is, the actually-measured intensities of fluorescence werecorrected in accordance with the following formula (1):f _(n) =f _(hyb,n) /f _(den,n)  (1)where

-   -   f_(n): correction value for the intensity of fluorescence in        each cycle,    -   f_(hyb,n): the intensity of fluorescence at 72° C. in each        cycle, and    -   f_(den,n): the intensity of fluorescence at 95° C.: in each        cycle.

It is FIG. 15 that was obtained by plotting the thus-obtained valuesagainst the corresponding cycles.

(b) A processing step that the values correction-processed by formula(1) in the respective cycles were introduced into the formula (3) tocalculate the rates of changes (decreases or quenches, %) influorescence between the samples in the respective cycles, namely, astep for performing processing in accordance with the following formula(10):F _(n) =f _(n) /f ₂₅  (10)where

-   -   F_(n): processed value of each cycle,    -   f_(n): value of each cycle as obtained in accordance with        formula (1), and    -   f₂₅: value of the 25^(th) cycle as obtained in accordance with        formula (1)

Formula (10) is similar to formula 3 except for a=25.

(d) A step that the processed value of each cycle as obtained in step(b) was subjected to processing in accordance with formula (6) to obtainthe logarithm of the rate of a change (decrease or quench, %) influorescence intensity, namely, a step for performing processing inaccordance with the following formula (11):log {(1−F _(n))×100}  (11)where

-   -   F_(n): value obtained in accordance with formula (10).

Formula (11) is similar to formula (6) except for b=10 and A=100.

The above results are shown in FIGS. 16 and 17.

FIG. 16 is a print-out obtained by plotting the values, which have beenprocessed in steps (a) and (b), against the corresponding cycles.

FIG. 17 is a print-out obtained by processing the values, which havebeen obtained in FIG. 16, in a similar manner as in step (d) and thenplotting the thus processed values against the corresponding cycles.

Next, based on the graph of FIG. 17, processing was performed throughsteps (f), (g) and (h). Described specifically, as in ComparativeExample 1, 0.1, 0.3, 0.5, 0.7, 0.9 and 1.2 were chosen as thresholds forlog (rates of changes in fluorescence intensity, %) The numbers ofcycles in which the logarithums reached the thresholds were plottedalong X-axis, while the numbers of copies of the human genome DNA beforethe initiation of reactions were plotted along Y-axis, whereby workinglines were drawn. The results are shown in FIG. 18. Correlationcoefficients (R²) determined by conducting processing in steps (i) and(j) with respect to those working lines were 0.998, 0.999, 0.9993,0.9985, 0.9989 and 0.9988, respetively. From those correlationcoefficients, it was able to confirm that adoption of 0.5 as a threshold(correlation coefficient: 0.9993) is desired. It is understood that witha working line having this correlation coefficient, the number of copiesbefore initiation of a reaction can be accurately determined with respetto a nucleic acid sample the number of copies of which is unknown.

EXAMPLE 23

This Example is directed to an analysis of a melting curve of a nucleicacid and also to an analysis of a Tm value.

A software comprising the following steps was created: 1) with respectto a nucleic acid amplified by the novel PCT method of the presentinvention, gradually heating the amplified nucleic acid from a lowtemperature until the nucleic acid is completely denatured (for example,from 50° C. to 95° C.), or gradually lowering it; 2) in step 1),measuring the intensity of fluorescence at short time intervals (forexample, at intervals equivalent to temperature rises of from 0.2° C. to0.5° C.); 3) displaying the measurement results of step 2) on a displayas a function of time, namely, displaying a melting curve of the nucleicacid; 4) differentiating the melting curve obtained in step 3); 5)displaying, on a display, derivatives (−dF/dT, F: fluorescenceintensity, T: time) obtained in step 4); and 6) determining a point ofinflection from the derivatives obtained in step 5). Using “LightCycler™System” (manufactured by Roche Diagnostics, Mannheim Germany) in which acomputer-readable recording medium with the data analysis softwarerecorded therein had been installed, the novel real-time quantitativePCR reaction of the present invention was conducted to analyze themelting curve of the nucleic acid. In the present invention, theintensity of fluorescence increases with the temperature.

With respect to 1 copy and 10 copies of the same human genome DNA as inExample 22, PCR was conducted in a similar manner as in Example 20. FIG.19 is a print-out of data obtained by processing data of the PCR insteps 1), 2), 3), 4) and 5). Concerning 75^(th) amplification productsof the 1 copy and 10 copies, data were processed in steps 1), 2 and 3)of this Example. The nucleic acid melting curves so obtained are shownin FIG. 20. Those curves were differentiated in step 4), and points ofinflection (Tm values) were determined in step 5) and 6). Thedifferentiated curves with the points of inflection are ilustrated inFIG. 21. It was ascertained from FIG. 21 that the amplification productsof the 1 copy and 10 copies were different products as their Tm valueswere different from each other.

1. A method for monitoring PCR amplification of a target nucleic acid,which comprises: conducting a PCR reaction with a forward primer, areverse primer, and a nucleic acid probe which is labeled with afluorescent dye, wherein only said nucleic acid probe is provided forone type of target nucleic acid, wherein said probe is labeled at an endportion thereof with said fluorescent dye, and said probe has a basesequence designed such that, when said probe is hybridized with saidtarget nucleic acid, at least one G (guanine) base exists in a basesequence of said target nucleic acid at a position 1 to 3 bases apartfrom an end base portion where said probe and said target nucleic acidare hybridized with each other; wherein said fluorescent dye is reducedin fluorescence emission when said probe is hybridized with said targetnucleic acid or amplified target nucleic acid; and monitoring atime-course of said amplification of the target nucleic acid bymeasuring a fluorescence intensity in said PCR reaction.
 2. The methodaccording to claim 1, further comprising adding, before saidhybridization, a helper probe to a reaction system in which saidhybridization is conducted.
 3. The method according to claim 1, whereinsaid nucleic acid probe and said target nucleic acid are hybridizedafter said target nucleic acid is subjected to heat treatment underconditions suitable for sufficient degradation of a higher-orderstructure of said target nucleic acid.
 4. The method according to claim1, wherein said probe has G or C as a 3′ end base and is labeled at said3′ end thereof with said fluorescent dye.
 5. The method according toclaim 1, wherein the nucleic acid probe has G or C as a 5′ end base andis labeled at said 5′ end thereof with said fluorescent dye.
 6. Themethod according to claim 1, wherein a hydroxyl group of a 2′ or 3′carbon of a ribose or a 3′ carbon of a deoxyribose at 3′ end of saidprobe has been phosphorylated.
 7. The method according to claim 1,wherein an oligoribonucleotide of said probe is a chemically-modifiednucleic acid.
 8. The method according to claim 1, wherein anoligonucleotide of said probe is a chimeric oligonucleotide comprising aribonucleotide and a deoxyribonucleotide.
 9. The method according toclaim 8, wherein said ribonucleotide is a2′-O-methyloligoribonucleotide.
 10. The method according to claim 1,wherein said fluorescent dye is selected from the group consisting ofFITC, BODIPY FL, BODIPY FL/C3, 6-joe, BODIPY TMR, BODIPY FL/C6, Alexa488, and Alexa
 532. 11. The method according to claim 1, wherein saidtarget nucleic acid is RNA.
 12. The method according to claim 1, whereinsaid target nucleic acid is a nucleic acid purified from a microorganismor animal.
 13. The method according to claim 1, wherein said targetnucleic acid is in cultivated cells of a microorganism or in ahomogenate of cells.
 14. A method for monitoring PCR amplification of atarget nucleic acid, which comprises: conducing a PCR amplificationreaction of the nucleic acid with a forward primer and a reverse primer,wherein the forward primer and the reverse primer are provided for onlyone type of target nucleic acid, wherein at least one of the forwardprimer and the reverse primer is labeled at a 5′-end portion thereofwith a flourescent dye, and has a base sequence designed such that, whenthe at least one of the forward primer and the reverse primer arehybridized with the target nucleic acid, at least one guanine baseexists in a base sequence of the target nucleic acid at a position 1 to3 bases apart from an end base portion where the labeled at least one ofthe forward primer and the reverse primer and the target nucleic acidare hybridized to each other, wherein the fluorescent dye is reduced influorescent emission when the labeled at least one of the forwardprimer, the reverse primer, the forward primer extended by the PCRamplification, and the reverse primer extended by PCR amplification ishybridized to the nucleic acid; and monitoring a time-course of the PCRamplification of the nucleic acid by measuring an intensity offluorescence in the PCR amplification reaction.
 15. A method fordetermining an initial concentration of a nucleic acid before PCRamplification of the nucleic acid, which comprises conducting thereal-time monitoring of PCR amplification according to claim 14;measuring a fluorescence intensity of the PCR reaction in each cycle inwhich the the labeled at least one of the forward primer, the reverseprimer, the forward primer extended by the PCR amplification, and thereverse primer extended by PCR amplification; and the target nucleicacid or amplified target nucleic acid have hybridized with each other;detecting a threshold cycle number (a Ct value) at which a decrease influorescence intensity of the PCR reaction in each cycle in which thelabeled at least one of the forward primer and the reverse primer; andthe target nucleic acid or amplified nucleic acid hybridized has beenobserved; and correlating the Ct value to an initial concentration ofthe target nucleic acid before the PCR amplification.
 16. The methodaccording to claim 14, wherein at least one of the forward primer andthe reverse primer is labeled with a fluorescent dye on a cytosinenucleotide; measuring a fluorescence intensity of the PCR reaction ineach cycle in which the labeled at least one of the forward primer, thereverse primer, the forward primer extended by the PCR amplification,and the reverse primer extended by PCR amplification the probe and thetarget nucleic acid or amplified target nucleic acid have hybridizedwith each other; detecting a threshold cycle number (a Ct value) atwhich a decrease in fluorescent intensity of the PCR reaction in eachcycle in which the labeled at least one of the forward primer, thereverse primer, the forward primer extended by the PCR amplification,and the reverse primer extended by PCR amplification and the targetnucleic acid or amplified nucleic acid hybridized has been observed; andcorrelating the Ct value to an initial concentration of the targetnucleic acid before the PCR amplification.
 17. The method according toclaim 14, wherein a hydroxyl group of a 2′ or 3′ carbon of a ribose or a3′ carbon of a deoxyribose at 3′ end of one of the forward primer andthe reverse primer has been phosphorylated.
 18. The method according toclaim 14, wherein an oligoribonucleotide of one of the forward primerand the reverse primer is a chemically-modified nucleic acid.
 19. Themethod according to claim 14, wherein an oligonucleotide of one of theforward primer and the reverse primer is a chimeric oligonucleotidecomprising a ribonucleotide and a deoxyribonucleotide.
 20. The methodaccording to claim 19, wherein said ribonucleotide is a2′-β-methyloligoribonucleotide.
 21. The method according to claim 14,wherein said fluorescent dye is selected from the group consisting ofFITC, BODIPY FL, BODIPY FL/C3, 6-joe, BODIPY TMR, BODIPY FL/C6, Alexa488, and Alexa
 532. 22. The method according to claim 14 wherein saidnucleic acid is RNA.
 23. The method according to claim 14, wherein saidnucleic acid is a nucleic acid purified from a microorganism or animal.24. The method according to claim 14, wherein said nucleic acid is incultivated cells of a microorganism or in a homogenate of cells.